close

Вход

Забыли?

вход по аккаунту

?

Isotope Effects in Organic Chemistry and Biochemistry.

код для вставкиСкачать
the Me-Vg antigen. The results further indicate for both
antigens destruction of part of the carbohydrates by a n additional “peeling reaction” since approximately twice as many
sugar residues as amino acid residues are destroyed in the N N
antigen; the proportion is even 5 :1 for the Me-Vg antigen [961.
It can be concluded from the sum of the chemical and biological results that the sugar chains have a length of only 4-6
monosaccharides in the NN antigen bdt that they must be
considerably larger in the meconium antigen. The immunological specificity is probably determined by small oligosaccharide groupings on these chains : multiple units of not more
than 2 sugars for Viciu specificity and not more than 4 for
human N specificity (measured with the Armstrong anti-N
serum). The total number of chains with N specificity as
determined with human anti-N serum is at most approximately 300 per molecule of antigen Ca 825 [961.
The MM and N N antigens possess more than one biological
function. Therefore they can serve as models not only in
blood-group serology, biochemical genetics of man and in
virology but in a number of other fields. The N N antigen is
the first physically homogeneous, chemically defined cell
membrane component of a mammal which possesses bloodgroup activity of the same order as that of the best ABH(0)
glycoproteins,determinedwith their corresponding antibodies,
but isolated from body secretions.
Received: March 17th, 1966
[A 535 IE]
German version: Angew. Chem. 78,967 (1966)
Translated by Express Translation Service, London
Isotope Effects in Organic Chemistry and Biochemistry
BY PROF. H. SIMON AND DR. D. PALM
CHEMISCHES INSTITUT DER FAKULTAT FtfR LANDWIRTSCHAFT UND GARTENBAU,
WEIHENSTEPHAN, AND ORGANISCH-CHEMISCHES INSTITUT DER TECHNISCHEN HOCHSCHULE
MUNCHEN (GERMANY)
The present article shows the extent 10 which isotope effects are likely to be encountered in
the use of isotope techniques and what problems are studied with primary arid secondary
isotope effects. By way of example, the results of studies on E2 reactions, particularly in the
Hofmann degradation, are discussed, foilowed by a discussion of some “analytical isotope
effects”. Finally, the problems encountered and the information that can be obtained from
isotope effect studies on enzyme reactions, and the advantages and disadvantages of coinpetitive and non-competitive techniques are described. In addition to a survey of isotope
efsects in dehydrogenase reactions, new isotope efsects encountered in the dehydrogenation
of T-labeled alcohols are reported.
I. Introduction
When stable or radioactive isotopes are used as tracers
in organic chemistry and biochemistry, it is usually
assumed that the various nuclides of an element behave
in the same way, i.e., that there is no discrimination
between labeled and unlabeled molecules in chemical
or physical operations. However, quantitative investigation shows that some such discrimination does exist.
These isotope effects [1-9al are due mainly to differences
[I] J. Bigeleisen and M. G. Mayer, J. chem. Physics 15, 261
(1947).
[2] S. S. Roginski: Theoretische Grundlagen der Isotopenchemie. VEB Deutscher Verlag der Wissenschaften, Berlin 1962,
pp. 1-107.
[ 3 ] J. Bigeleisen and M . Wolfsberg: Advances in Chemical
Physics, Vol. I. Interscience, New York 1958.
[4] L. Melander: Isotope Effects on Reaction Rates. Ronald
Press, New York 1960.
[5] C . J. Collins in V . Gold: Advances in Physical Organic Chemistry, Vol. 11. Academic Press, New York 1964, p. 60.
[6] H . Zollinger in V . Gold: Advances in Physical Organic Chemistry, Vol. 11. Academic Press, New York 1964, p. 163; Angew.
Chem. 70, 204 (1958).
[ 7 ] P . Buertschi, Z. naturwiss. med. Grundlagenforsch. 2, 22
(1964).
[8] E. A. Halevi in S. G. Cohen, A. Streitwieser jr., and R . W.
Tuft: Progress in Physical Organic Chemistry, Vol. I. Interscience,
New York 1964, pp. 109-221.
920
in the masses of the nuclides and the resulting differences
in the zero-point energies of the bonds (cf. Fig. 1).
Some (very small) isotope effects are due to other properties of the nuclei, such as the quadrupole moment and
the magnetic moment.
Mean X - H distance
Mean X - 0 distance
@m
Internuclear distance +
Fig. I Potential energy of a molecule HX and of the transition state.
The asymmetry of the potential energy curve (exaggerated for clarity)
results in a slightly shorter equilibrium internuclear distance in D X than
in HX. The different zero-point energies E g and E g lead to different
activation energies for reaclions of HX and DX (arrows) and are the
principal cause of the kinetic isotope effect.
The smaller the relative difference between the masses
o f two nuclides, the less pronounced are the isotope
effects. Thus already in the carbon isotopes the isotope
Angew. Chem. internat. Edit.
Vol. 5 (1966)
No. 11
effects generally are not very important. On the other
hand, isotope effects must always be taken into account
in the use of the hydrogen isotopes, since often experimental results can be interpreted only in this way,
particularly in a reaction in which bonds formed by the
hydrogen isotope itself or adjacent bonds are broken.
The main rcasons for the publication of more than 100
original papers and several reviews t5-81 on isotope
effects alone in 1964 are:
1. The so-called kinetic isotope eflect can be used in the
study of reaction mechanisms.
According to the theory of absolute reaction rates [9a,hl, the
kinetic isotope effect is equal to the quotient of the equilibrium constants for the formation of the transition states in the
unlabeled and labeled compounds, respectively (cf. the discussion of equilibrium isotope effects under 2 below). The
calculation is therefore possible when the vibrational frequencies of the ground state and of the transition state of the
reactants are known[3,4,9,9a, 101. The frequencies in the
transition state are unknown; the frequencies in the ground
state and the isotope effect, however, can be measured. One
can therefore obtain information about the transition state
in this way. Wolfsberg and Stern"o1 and Willi[ll-131 calculated theoretical isotope effects for concrete transition states
on the basis of the ideas developed by Bigeleisen and others
C1-41. Computer programs have been developed for the calculation of the influence of various parameters o n the isotope
effects [ l o , 10aI.
The problems most frequently studied in current experimental work are that of the rate-determining step of a sequence of reactions and that of the extent to which a bond
has already been broken or formed in the transition state.
2. The equilibrium isotope efects observed in exchange
equilibria are of industrial importance in isotope separation. For example for the exchange reaction used for
the production of heavy water 1141,
H20+ HDS
kz
HDO+ H2S
the equilibrium constant at 25 "C is kllk2 = K = 2.2
(this is an extreme value, most values being nearer to 1).
The knowledge or estimation of equilibrium isotope
effects also is of interest in the preparation ofcompounds
labeled with hydrogen isotopes and in biochemical
studies. In biochemical reaction sequences, e.g. glycolysis, proceeding in tritiated water it is found that for
reversible reaction steps resulting in equilibration, the
hydrogen isotope content of a given position in the
intermediates differs from the isotope content of the
water only because of an equilibrium isotope effect.
However, if reversibility is incomplete, the isotope
[9] J. Bigeleisen, Pure appl. Chem. 8, 217 (1964).
[9a] K . B. Wiberg: Physical Organic Chemistry. Wiley, New
York-London-Sydney 1964.
[9b] A. A . Frost and R. G. Pearson: Kinetics and Mechanism.
Wiley, New York 1961.
[lo] M. Wolfiberg and M . J. Stern, Pure appl. Chem. 8, 225
(1964).
[lOa] J. H. Schachtschneider and R . G. Snyder, Spectrochim.
Acta 19, 117 (1963).
[ll] A . V. WiNi, Helv. chim. Acta 47, 647 (1964).
[12] A. V. WiNi, Helv. chim. Acta 47, 655, 837 (1964).
1131 A . V. Willi, Ber. Bunsenges. physik. Chem. 63, 266 (1964).
[14] H . C. Urey, J. chem. SOC. (London) 1947, 562.
Angew. Chem. internat. Edit.
/ Vol. 5 (1966) / No. I 1
contents are smaller, since the splitting of a water molecule always iwolves an intramolecular isotope effect,
which frequently amounts to about 3.5-4 in the case of
HOD and about 7 in the case of HOT (cf. Section V.4).
The equilibrium constant for the distribution of an isotope
between two molecules A and B in the reaction A + B' +
A'
B is given by
+
where
n = number of degrees of fresdom
Qi
partition function of i-th molecule in the ground state
hvIkT
= Planck's constant
= frequencies of the normal modes
= Boltzmann's constant
= absolute temperature.
=
u =
h
v
k
T
At the temperatures normally encountered in organic chemistry and biochemistry, the equation
holds approximately. For the complete calculation of examples see [9a, 14al.
There is a close relation between equilibrium isotope effects
and reaction mechanisms. Thus the change in rate constants
when DzO is used instead of H20 as solvent for acid- or basecatalysed reactions is often used to distinguish between different mechanisms [15-20al. However, definite conclusions can
be drawn only if the order of magnitude of the isotope effect
for a certain mechanism can be calculated theoretically.
Equilibrium isotope effects are important in geology
also [21 s 221.
3. As will be shown in Section V, isotope effects are
very important in biochemistry, and will contribute
further to a deeper insight into biochemical mechanisms.
4. So-called "analytical isotope effects" are observed in
analytical tracer studies. Labeled compounds generally
can be used only if they can be purified to colistant
specific radioactivity by recrystallization, distillation, or
a continuous distributioii method. Owing to isotope
effects, this is sometimes impossible because of incomplete establishment of exchange equilibria and
kinetic effects.
[14a] J. C. Evans and G. Y. S. Lo, J. Amer. chem. SOC.88, 2118
(1966).
[14b] J . K . Wiltnshurst and H . J. Bernstein, Canad. J. Chem. 35,
226 (1957).
[15] R. P. Bell: Acid-Base Catalysis. Oxford University Press,
London 1941.
[16] K . B. Wiberg, Chem. Reviews 55, 713 (1955).
[17] F. A . Long and J. Bigeleisen, Trans. Faraday SOC.55, 2077
(1959).
[18] A . V. WiNi, Z . Naturforsch. 196, 461 (1964).
[19] A . V. WiNi: Saurekatalytische Reaktionen der organischen
Chemie. Vieweg, Braunschweig 1965.
[20] A. J. Kresge, Pure appl. Chem. 8, 243 (1964).
[20a] H . H . Huang, R . R . Robinson, and F. A . Long, J. Amer.
chem. SOC.88, 1866 (1966).
1211 H . C. Urey, H. A . Lowenstam, S. Epstein, and C. R . McKinney, Bull. geol. SOC.America 62, 399 (1951).
[22] P . Baertschi, Schweiz. mineralog. petrogr. Mitt. 37, 73
(1957).
921
II. Primary a n d Secondary Kinetic Isotope Effects
1. Primary Isotope Effects
According to the theory of absolute reaction rates [9a99bJ,
the maximum kinetic isotope effect at a given temperature
is obtained when the bond is completely broken in the
transition state. However, in the removal of a proton
from a carbon atom by a base, for example, the proton
becomes bonded to the base B as it is removed from the
carbon. The maximum isotope effect therefore occurs
at the point of "half-separation'' C...H...B . If, on the
other hand, the proton in the transition state is more
strongly bonded to the C atom (C..H..*.B), or to the
base (C....H..B), the isotope effects are smaller.
The mathematical treatment of this effect is given by Westheimer 1231 and by Bigeleisen [91. On the basis of theoretical
considerations and calculations, however, Willi and Wolfsconcluded that a pronounced maximum of the isoberg
tope effect as a function of the breaking and formation of
bonds in the transition state depends o n the form of the
potential barrier separating the reactants from the products.
No experiments confirming this view appear t o have been
published.
ation of deuterated 2,4-dibronio-l,3,5-trimethoxy.
benzene [*6el. In oxidations [*7-321, on the other hand,
the hydrogen isotope effects are usually close to the
maximum value.
Isotope effects for tritium are often calculated from
those for deuterium with the aid of the equation [251
(but cf. also 1261).
In addition to the relatively large isotope effects o f t h e
hydrogen isotopes, many effects with the nuclides W ,
14C, 15N 180, and 34s or 3 5 s have been measured 141.
Values of up to 1.086 at 11 "C
have been reported
for k12clk13~in S N reactions;
~
in s N 1 reactions, on
the other hand, the isotope effects are much smaller, and
sometimes even inverted [*6bl. The effects for 12C/14C
may be assumed to be twice as large. In the decarboxylation of [carboxy-14C]benzoic acid and its derivatives, isotope erects of 1.068 to 1.78 have been observed
at 210 and 238"C[26cJ, whereas the maximum value
expected was 1.057131. Most of the values foucd for
k 1 4 ~ / k 1 5are
~ less than 1.03.
In reactions involving several stages. isotope effects can
naturally be observed only if the bond to the isotope is
broken in the rate-determining step (primary isotope
effect). The maximum isotope effect in the rupture of
carbon-hydrogen bonds is generally kH/kD [*I = 8.3 at
0 "c,kH/kD = 6.9 at 25 "c, etc. f4J. However, this is
true only if the proton transfer takes place without
tunnelling [24al.
Other types of reactions in which primary isotope effects have
been measured for the nuclides D,T, 13C, 14C, 15N, and 3 4 s
are solvolysis, decarboxylation, elimination, fragmentation,
and isomerization [41. A review has also been published [32al
on isotope effects in the unimolecular decomposition of
simple alkanes and alkyl radicals. An example of isotope
effects in metal-catalysed reactions is the decomposition of
deuterated formic acids [32bl.
In the 2,6-lutidine-catalysed iodination of [2-D]-2nitropropane for example, it was found that kH/kD =
24 at 25 "C, whereas in the same reaction with pyridine
as catalyst kH/kD = 9.8 [241. It was concluded that
stei ically hindered slow proton transfers can in general
exhibit extremely large isotope effects. The electrophilic
aromatic substitution of D or T generally exhibits only
slight kinetic isotope effects or none at all 161. However,
larger values can occur under certain steric conditions.
In the bromination of 1,3,5-tri-t-butyI[?-D]benzenein
the presence of silver ions it was found that kH/kD 3
3.6 [26dl, and a value of 4.75 was obtained in the bromin-
2. Secondary Isotope Effects
[23] F. H . Westheimer, Chem. Reviews 61, 265 (1961).
[23a] A . V. Willi and M. Wolfsberg, Chem. and Ind. 1964, 2097.
[*I Kinetic isotope effects generally are given as a ratio of rate
constants, the subscript denoting the nature of the nuclide.
[24] L . Funderburk and E. S. Lewis, J. Amer. chem. SOC.86, 2531
(1964).
[24a] V. J. Shiner jr., and B. Martin, Pure appl. Chem. 8, 371
(1964).
[25] C. G. Swain, E. C. Stivers, J. F. Reuwerjr., and L . J. Schaad,
J. Amer. chem. SOC.80, 5885 (1958).
[26] J. Bigeleisen in: Tritium in the Physical and Biological
Sciences, Vol. I. Internat. Atomic Energy Agency, Vienna 1962,
p. 161.
[26a] K . R . Lynn and P . E. Yankwich, J. Amer. chem. SOC.83,
790, 3220 (1961).
[26b] A . J. Kresge, N . N.Lichtin, and K . N. Rao, J. Amer. chem.
SOC.85, 1210 (1963).
[26c] M . Roessler and H . Koch, Radiochim. Acta 4, 12 (1965).
[26d] G. Illuminati and F. Stegel, Ric. sci., Parte 11, Sez. A 7, 460
(1964); Chern. Abstr. 63, 5466c (1965).
Detailed and critical treatments and reviews of the
secondary isotope effect have been published by
Halevi[*l and others [331. In the secondary isotope effect,
no bond to the nuclide in question is broken in the ratedetermining step. Here also equilibrium and kinetic
isotope effects can be distinguished.
The difference in the dissociation constants of
C6H5-CH2-COOH and C6H5-CD2-COOH (KH/KD =
1.12) is 9 secondary equilibrium isotope effect. Similarly, CsH5-CD2-NH2
is a stronger base than
____
[26e] E. Helgstrand, Acta chem. scand. 19, 1583 (1965).
[27] W.P. Jencks in A . F. Scott: Survey of Progress in Chemistry,
Vol. I. Academic Press, New York 1963.
[28] R . Stewart: Oxidation Mechanisms: Application to Organic
Chemistry. Benjamin, New York-Amsterdam 1964.
[29] K. B. Wiberg and P . A . Lepse, J. Amer. chem. SOC.86, 2612
(1964).
[30] 0. H . Wheeler, Canad. J. Chem. 42, 706 (1964).
[31] D . G. Hoare and W. A . Waters, J. chem. SOC.(London) 1964,
2560.
[32] A . J. Green, T . J. Kemp, J. S. Littler, and W . A . Waters,
J. chem. SOC.(London) 1964, 2722.
[32a] B. S . Rabinovitch and D. W . Setser in W . A . Noyes jr.,
G. S . Hammond, and J. N . Pitts j r . : Advances in Photochemistry,
Vol. 111. Interscience, New York 1964, pp. 1-82.
[32b] G. M . Schwab and A . M . Watson, Trans. Faraday SOC.60,
1833 (1964).
[33] R. E. Weston jr. in: Annual Reviews of Nuclear Science.
Annual Reviews, Inc., Palo Alto 1961, p. 439.
Angew. Chem. internat. Edit.
1 VoI. 5 (1966) 1 No. II
C6Hs-CH2-NH2 (K,"/K," = 1.13) 1341. These values
appear to be uncertain, and isotope effects of 1.06[341 to
1.03 1344 have been reported for CH3COOH and
CD3COOH. On the other hand, the differences in the
rates of the Hofmann degradation of
(CH3)3N@CaHT-CpH2-CYH3 OH@
(1)
(CH3)3N@-CaH2-CBHT-CYH3 OH"
(2)
(if HOH, and not HOT, is eliminated), and
(CH3)3N@-CaH2-CPH2-CyHlT
OH"
(3)
Hydrogen isotope substitution affects dipole moments,
nuclear quadrupole coupling constants, and optical
activity. From the slight anharmonicity of the molecular
energy function (Morse function) [9b], it is possible (cf.
Fig. 1) to derive a small difference in electron distribution between C-H and C-D bonds, resulting in slight
bond shortening and slightly stronger electron donation
by a C-D bond than by a C-H bond 1x1.
The dipole moments of ND3, CD3ND2, DCNO, and
(CH3)3CD are 0.01 to 0.04 Debye larger than those of
the corresponding hydrogen compounds [38-38b]. It can
therefore be stated that a C--D bond exerts a stronger
inductive effect than a C-H bond. Thus CsHs-CDz@I
in relation to the unlabeled compound are secondary
isotope effects. In the case of ( I ) , a so-called a-isotope
effect occurs in the breaking of the C-N bond, and a
P-isotope effect in the breaking of the Cp-H bond; etc.
Following Streitwieser [34bl, Halevi [81 distinguishes
between secondary isotope effects of the first and second
kind. The criterion for this distinction is whether or not
the bond to the isotope changes its steric orientation as
a result of the reaction. For example, the transition of
a deuterated C atoni from sp3 to sp2 hybridization is an
effect of the first kind, as is the Hofmann degradation
of ( I ) and ( 2 ) ; the degradation of (3), on the other
hand, is an effect of the second kind.
Since a methyl group, for example, often is completely
deuterated, the isotope effects are generally referred t o 1 D
(e.g., A A F = (RTjn)ln ( k H / k D ) , where n is the number of D
atoms per molecule).
Collins 1351 also found an example of a secondary 14C isotope
effect in the reaction of CaH5-14CO-CH3 with 2,4-dinitrophenylhydrazine ( k 1 2 c l k 1 4 ~= 1.0085 0.0004). However,
it is doubtful whether any conclusions can be drawn on the
basis o f such small effects.
The theories on the secondary isotope effect are involved
and in part contradictory [8,331. Therefore we shall give
only a few physical facts that are closely related to the
chemical reactivity, and frequently confirmed rules on
the direction and magnitude of the effects in various
types of reactions.
There are fundamentally two views:
1. Secondary isotope effects may be regarded as substituent effects, a C-D bond having a stronger +1 effect
than a C-H bond[s,lo,*3,33,34,36,371
and a CD3 group
exhibiting weaker hyperconjugation than a CH3
group 1531.
2. Secondary hydrogen isotope effects can be explained
by the smaller bulk of deuterium 133a1, i.e. by the smaller
amplitude of vibration of D as compared with H[411.
[33a] H . C . Brown and G . J. McDonald, J. Amer. chem. SOC.88,
2514 (1966); H. C . Brown, M . E. Azzaro, J. G . Koelling, and G. J.
MeDcnald, ibid. 88, 2520 (1960).
[34] E. A. Halevi and M. Nussim, Bull. Res. Council Israel Sect.
A 5, 263 (1956); J. chem. SOC.(London) 1963, 866.
[34a] M . Paabo, R . G. Bates, and R . A . Robinson, J. physic.
Chem. 70, 540 (1966).
[34b] A . Streitwieserjr., R. H . Jagow, R. C. Fahey, and S. Suzuki,
J. Amer. chem. SOC.80, 2326 (1958).
[35] V. F. Raaen, A . K . Tsiomis, and C . J. Collins, J. Amer.
chem. SOC.82, 5502 (1960).
1361 A. Streitwieserjr., Ann. New York Acad. Sci. 84, 576 (1960).
[37] M. Wolfsberg, J. chem. Physics 33, 2 (1960).
Angew. Chem. internat. Edit.
1 Vol. 5 (1966) / No. I I
COOH and C6H5-CD2- NH3 are less dissociated than
the undeuterated compounds. The influence of deuterium becomes particularly clear in a comparison of the
nuclear quadrupole coupling constants of CH3X and
CD3X (X = halogen). The C-X bonds are more
strongly ionic in the deuterated compounds.
The same conclusion is reached by a study of the effect
of deuteration on the chemical shifts of nuclear magnetic
resonance signals. The 1H signals of -CH2D are displaced by 0.015 ppm relative to those of -CH3 [391, and
the 19F signals of -CF2D by 0.60 5- 0.05 ppm relative
to those of - C S H 1401. However, in a recent comparison
of rn-fluorotoluene and [methyl-D3]-m-fluorotoluene,
the expected shift of the 19F signal was not observed [4Oa1.
Electron spin resonance measurements on deuterated
benzene, naphthalene, and cyclooctatetraene also failed
to show any stronger electron-donating action of
deuterium [40dl.
The C-D bond is less polarizable than the C-H bond;
this property results in optical asymmetry in compounds
of the type RIRZCHD. A theory developed to explain
the optical activity due to isotope effects is based on
the interaction of light with the various atoms
and isotopes 140cl. Streitwiesar [361 refers to the similarity of the optical rotatory dispersion curves of
CH3-CH2-CH2-CHD(OH),
CH3-CHD(OH), and
CH3(CH2)sCH(OH)-CH3 [40b].
It is concluded from these facts that this "electronic
isotope effect" [*I may be treated as a true substituent
effect. The effect of replacement of a hydrogen atom in
a C-H bond by D is qualitatively similar to that of
replacement by a CH3 group. The replacement of a
CH3 by a CD3 group has a similar effect to that of
replacement by C2H5 or even by (CH3)3C.
[38] J . M . A . DeBruyne and C . P . Smyth, J. Amer. chem. SOC.
57, 1203 (1935).
[38a] D . R. Lide, J. chem. Physics 27, 343 (1957); 33,1519 (1960).
[38b] J. N . Shoolery, R. G. Shulman, and D . M . Jost, J. chem.
Physics 21, 1416 (1953).
[39] G. van Dyke Tiers, J. chem. Physics 29, 963 (1958).
[40] G. van Dyke Tiers, J. Amer. chem. SOC. 79, 5585 (1957).
[40a] G. E. Maciel and D . D. Traficante, J. Amer. chem. SOC.87,
2508 (1965).
[40bl A. Streitwiestr jr., L. Verbit, and S . Andreades, J. org.
Chemistry 30, 2078 (1965).
[ ~ O C ] N . V. Cohan and H . F. Hameka, J. Amer. chem. SOC.88,
2136 (1966).
[40d] M . Karplus, R. G. Lawler, and G . K . Fraenkel, J. Amer.
chem. SOC.87, 5260 (1965).
923
The “transmission” of isotope effects via multiple bonds
and benzene nuclei has been discussed by Shiner and
Kriz 1531.
The effects observed on replacement of H by D or T can
be explained in another way: BartelIr411 believes that
bDth the a- and the P-deuterium isotope effects are due
to the greater amplitude of vibration of C-H bonds as
compared with C-D or C-T bonds. The repulsive
forces between H atonis are consequently greater than
those between H atoms and deuterium or tritium, or
between deuterium atoms or tritium atoms. Moreover,
the repulsion between non-bonded atoms fixed to an
sp3 carbon atom is greater than that between atoms
attached to C atoms in the sp2 state. Consequently, the
energy gain resulting from the decrease in repulsive
forces in going from sp3 to sp2 hybridization is greater
in protium compounds than in deuterium or tritium
compounds. It may be deduced from this that in a transition from the sp3 to the sp2 state, H compounds will
react faster than D or T compounds; this is a well
established rule [36342-461. The reverse is true in transitions from the sp2 to the sp3 staters,46,47J.
This has also been confirmed for a free-radical reaction.
In the polymerization of styrene, [a-D]-, [?$-D2]-,
[%-TI-, and cr,P-trans-Tz-styrene react faster than unlabeled styrene [47aI.
The smaller bulk of CD3 groups as compared with CH3
groups has been clearly shown by H . C. Browiz[33al.
The quateriiization of 2-trideuteriomethylpyridine exhibits an inverse secondary isotope effec. of 0.97 with
methyl iodide, 0.96 with ethyl iodide, and 0.935 with
isopropyl iodide. The value found for 2,6-bis(trideuteriomethy1)pyridine was 0.92. Since 3-trideuteriomethylpyridine and 4-trideuteriomethylpyridine exhibit only
insignificant isotope effects, Brown considers any explanation of secondary isotope effects other than by
steric effects as unacceptable.
The same conclusion is suggested by the fact that only 2,6bis(trideuteriomethy1)pyridine has a greater heat of reaction
(by 0.23 kcal/mole) with BF3, than the undeuterated compound, and not the 3- and 4-methyl compounds.
Streitwieser et a/.[481 first demonstrated the influence of
D in the cr-position in s N 1 solvollses (transition state
with sp2 character), and interpreted it as being due to
the higher energy gain of the H compound on transition
from the sp3 to the sp2 state in the activated complex.
They described an approximation with which kH/kD
can be determined from the (geneidly known) vibration
[41] L . S. Bartell, J. Amer. chem. SOC.83, 3567 (1961).
[42] H . Simon and G. Heubach, Z . Naturforsch. 18b, 160 (1963).
[43] H . Simon and G. Mullhofer, Chem. Ber. 96, 3167 (1963).
[44] H . Simon and G . MuIlhofer, Pure appl. Chem. 8, 379 (1964);
lecture at the Symposium on Isotope Mass Effects in Chemistry
and Biology. Vienna, Dec. 9th-l3th, 1963.
[45] H . Simon and G. Miillhofer, Chem. Ber. 97, 2202 (1964).
1461 A . A . Zavitsas and S. Seltzer, J. Amer. chem. SOC.86, 3836
(1964).
[47] S . Seltzer, J. Amer. chem. SOC.83, 1861 (1961).
[47a] W . A . Pryor, R. W. Henderson, R. A . Patsiga, and N . Carroll, J. Amer. chem. SOC.88, 1199 (1966).
1481 A . Streitwieserjr., R. H. Jagow, R. C . Fahey, and S. Suzuki,
J. Amer. chem. SOC.80,2326 (1958).
924
frequencies of the unlabeled starting material and of the
product. Values of 1.12 to 1.15 are found for kH/kD of
many acyclic compounds, and somewhat higher values
for cyclic compounds [8,331.
Very small or even inverse isotope effects are observed
in S N reactions.
~
Thus we found1491 kH/kT = 0.96 for
the quaternization of pyridine with [Tlmethyl iodide
(see also [81). The solvolysis of halides and tosylates has
been intensively studied [50,51J; inverse isotope effects
were observed throughout this work. (Cf. also calculations by Willir131).
The possibility of using secondary isotope effects to obtain a better insight into the course of solvolysis reactions has been discussed by Lee and Wong[521. In the
solvolysis of [l,l-D2]-2-~yclopentylethyl,
[l,1-D2]-2-(2cyclopentenyl)ethyl, and [1,1-D2]-2-(3-~yclopentenyl)ethyl p-nitrobenzenesulfonates, an appreciable isotope
effect is observed only for the third compound, for
which kH/kD = 1.07 per D. It was concluded from this
~
result that the transition state has partial S Ncharacter,
and in agreement wich Streitwieser 1341, that neighboring
group effects are involved.
In the formation of methyl radicals from l-trideuteriomethyl-2-phenylethyl hypochlorite an a-deuterium isotope effect of 1.15 per D was observed, in good agreement with the results obtained in Sx1 reactions and
unimolecular thermal cleavages [461.
That the secondary isotope effect of kHlkD = 1.09 in
the hydrolysis of [1,1,1-D3]-4-chloro-4-methyl-2-pentyiie 1531 depends very little on the solvent is strong evidence that the hyperconjugation of a CD3 group is
weaker than that of a CH; group, since steric effects
cannot be involved in this case L531.
In addition to the much-studied s N 1 and S N ~
reactions [543, sxondary isotope effects have been used
to investigate Diels-Alder reactions [551. The results
suggest a four-center reaction with a transition state
situated largely on the reactant side.
Other steric isotope effects due to deuteration have been
observed in the racemization of 9,10-dihydro-4,5-dimethylphenanthrene 1561. Samples deuterated in the methyl
or methylene groups racemize more rapidlj . A distinct
isotope effect was also observed in the racemination of
2,2’-dibromobiphenyl-4,4‘-dicarboxylic
acid [571.
[49] H . Simon and D . Palm, Chem. Ber. 92, 2701 (1959).
[50] V. J. Shiner jr., H. R. Mahler, R. H. Baker j r . , and R. R .
Hiatt, Ann. New York Acad. Sci. 84, 583 (1960).
[51] K . T. Leffek, J. A . Llewellyn, and R. E. Robertson, Canad.
J. Chem. 38, 1505 (1960).
[52] C . C. Lee and E. W. C . Wong, Tetrahedron 21, 539 (1965).
[53] V. J. Shiner j r . and G. S. Kriz j r . , J. Amer. chem. SOC.86,
2643 (1964).
[54] See., e.g., A . Streitwieser jr. : Solvolytic Displacement Reactions. McGraw-Hill, New York 1962.
1551 D . E. Van Sickle and J. 0 . Rodin, J. Amer. chem. SOC.86,
3091
(1964).
[56] K . Mislow, R. Graeve, A . J. Gordon, and G. H. Wahl,jr., J.
Amer. chem. SOC.86, 1733 (1964).
[57] L . Melander and R. E. Carter, J. Amer. chem. SOC.86, 295
(1964).
Angew. Chem. internal. Edit. / Vol. 5 (1966)
1 No. I I
111. Isotope Effects in E 2 Reactions
such as the Hofmann Degradation
t h e strengthening of t h e bonds between t h e tertiary carbort
a n d t h e methyl groups i n t h e carbonium ionL6-31. As can be
seen, t h e tertiary butyl group exhibits no peculiarities in the
H o f man n degradation.
The study of isotope effects in E2 reactions is interesting
in that two bonds in the transition state are broken
“simultaneously”. One may therefore ask, e.g. in the
Hofmann degradation of quaternary cations of the type
(CH3)3N@-CaH2-CBH2-R‘, how this “simultaneous”
rupture depends on R , i.e. isotope effects can be used in
an attempt to establish whether the rupture of the C-N
bond or that of the Ce-H bond is faster. We therefore
determined the 14C and T isotope effects in the Hofmann degradation of the quaternary ammonium cations
listed in Table 1142-441.
k32slk34~for (CH&S@-C(CH3)3 is 1.007 [641. Th e 15N isotope
effect in t h e degradation of (CH3)3N @-CH2-CH2-C6H4-X-pP,
where X = H, C H 3 0 , a n d C1, does n o t vary with X [ 6 5 ] .
Whereas the isotope effects found for the rupture of the
C-N bond were mutually compatible and reasonable,
this was not so for the cleavage of the CP--H bond
when all known values were taken into account. The
tritium isotope effects found by us and the isotope effects from the literature are listed in Table 1. For ease
of comparison, all values are converted to 60 “C and the
deuterium values were converted to tritium values 1451.
Table 1. Summary of the primary 14C and T isotope effects (IEeXp)found by us [43-451, and comparison with
some D isotope effects found by various authors (recalculated for T) in the Hofmann degradation of quaternary ammonium cations ( C H ~ N Q R .
IEeXp, converted
to 40 “ C [a]
1.05 [b]
1.06[bl
1.073
1.034
1.064
1.038
IR
CHz-CHzT
CHZ-CH~T
CHz-CHT-CH3
CH2-CHT-crjHS
CH2-CHT-CsH4-NOz-p
CHz-CD,
CHz-cDl-caH~
IEexp. converted
to 60 “ C [a]
3.0 [a, b]
2.8 [a, bl
2.86 [a1
4.2 [a]
8 : 1.6[aI
125 4
14
3.9
[a] For experimental details, conversion OF the values found at various temperatures to 40 or 60 ”C, discussion
of error, e l c . , see [43-451
[b] Various conditions.
Though isotope effects were already known for the
breaking of C@-H bonds 158-601, the only value known
for the rupture of the C-Y bond (Y = N,S) was k32~/
k 3 4 ~= 1.0015 for the elimination of dimethyl sulfide
from P-phenethyldimethylsulfoniumbromide [611. Later
this value was revised to 1.0065 [61al.
h e y et al. found[621 a 15N isotope effect of 1.017
for (CH&N@-CHz-CH3 at 60°C and 1.009 for
(CH3)3Ne-CH2-CH2-C6H5.
The electron-attracting
phenyl group therefore decreases the isotope effect in
the rupture of the C-N bond. Our 14C isotope effects
(Table 1) are in good agreement with this conclusion
and showed even more clearly that the isotope effect is
increased by electron donors such as methyl groups
(cf. R = C3H7 with R = C2H5), and greatly decreased
by electron-attracting groups such as g-N02-C6H4.
The determination of t h e isotope effect of (CH3)3N@C(CH3)3
was of interest, since th e t-butyl group exhibits particularly
small isotope effects in SN1 reactions, presumably because of
[58] For elimination reactions see D. V. Banthorpe in E. D.
Hughes: Reaction Mechanisms in Organic Chemistry, Vol. 2.
Elimination Reactions. Elsevier, Amsterdam-New York 1963.
[59] W. H. Saunders j r . and D. H. Edison, J. Amer. chem. SOC.
82, 138 (1960).
[60] V. J. Shiner jr. and M. L. Smith, J. Amer. chem. SOC.80,
4095 (1958).
[61] W. H . Saunders j r . and S . Asperger, J. Amer. chem. SOC.79,
1612 (1957).
[61a] W. H . Saunders jr., A . F. Cockerill, S . Asperger, L. Klasine,
and D . Stefanovic, J. Amer. chem. SOC.88, 848 (1966).
[62] G. Ayrey, A. N . Bourns, and V. A . Vyas, Canad. J. Chem.
41, 1759 (1963).
Angew. Chem. internat. Edit. / Vol. 5 (1966) No. I I
It can be seen that the tritiuni isotope effect with
substituents in the P-position increases in the order
CH3 H < C6H5 < C6H4-N02-p[*].
<
As can be seen, a large hydrogen isotope effect in the
Hofmann degradation is accompanied by a small
carbon isotope effect, and vice versa. What does this tell
us about the transition state? For electron-attracting
groups, such as phenyl and p-nitrophenyl, the hydrogen
isotope effects are large, showing that the release of the
proton must have reached approximately the half-way
stage in the transition state (“half-release”, cf. Section
11.1). The question then arises of whether the release of
the proton in the presence of alkyl groups has progressed beyond or has not yet reached the “half-released”
state, since smaller isotope effects are observed in this
case.
[63] M . L. Bender and G. J. Buist, J. Amer. chem. SOC.80, 4304
(1958).
[64] W. H. Saunders j r . and S . E. Zimmermann, J. Amer. chem.
SOC.86, 3789 (1964).
[65] A. N. Bourns and P. J . Smith, Proc. chem. SOC.(London)
1964, 366.
[ * ] E. M. Hodnett and J. J. Sparapany, Pure appl. Chem. 8, 385
(1964), reported a much smaller value; however, this was not
determined directly as in the earlier measurements by Hodnett [66]
and by us [45], but was found indirectly from the kinetic and
secondary isotope effects. This procedure seems unsuitable in
view of the polymerization of p-nitrostyrene.
The value for (CH&N@-CH2-CD3 [60] deviates strongly
from this sequence. Since the primary object of these studies [60]
was not the determination of the isotope effects, a relatively
large error is possible: V. J . Shiner and M. L . Smith, persmal
communication.
925
A group R that facilitates the removal of a proton
(relative shift of the E2 elimination to a transition state
with carbanion character) may be assumed to hinder
the release of a leaving group that takes the electron
pair with it, and vice versa. It may also be assumed that
a negative charge on Cp cannot be stabilized by an
alkyl group, and that under the influence of the positive
charge on the nitrogen, the irreversible displacement of
electrons to form the products consequently occurs
already on slight loosening of the C@-H bond in the
transition state. In the case of a y l group, the negative
charge formed cail be better stabilized, so that the proton
can move farther away before the irreversible electron
transfer takes place.
chlorides, and the substitution of 3-chloro-1-butene by diethylamine. In this last case the 14C isotope effects for
[l-"K]-,[2-"T]-, and [3-14C]-3-chloro-l-butene
were found
to be 1.057 & 0.007, 1.074 + 0.003,and 1.079 i 0.003. The
chlorine isotope effect k35Cl/k37CI was found to be 1.0112 +
0.0006. This is a particularly good example, since it shows
that in the reaction
These assumptions are supported by the secondary isotope effects. If the transition state in the Hofmann
degradation were similar in structure to the final products, relatively large secondary isotope effects should
occur. However, as we have found for the quaternary
ammonium cations with R = CH2-CHT-CH3,
C H ~ - C H Z - C H ~ Tand
, CHT-CzH5, this is not so; we
obtained values of 1.33, 1.10, and 1.10 respectively [433.
IV. "Analytical" Isotope Effects
all the bonds of 3-chlorobutene-1 are influenced in the transition state.
If hydrogen-labeled compounds are subjected to simple
separations, e.g. recrystallization or distillation, which
are less efficient methods than multi-stage distribution,
surprisingly large isotope effects can nevertheless occur
when such a separation is associated with a chemical
reaction.
These conclusions are in disagreement with the assumpIn the recrystallization of [l-Tlmannose phenylhydraztion [67,681 that in the Hofmann degradation of quaterone from 60% aqueous methanol, for example, we
nary ammonium cations the rupture of the Cp-H bond
found that the labeled molecules crystallize more
is already relatively far advanced in the transition state.
slowly[70,711;kH/kr = 1.12. Owing to the importance
Since the isotope effect of the ethyl compound [601 (R =
of recrystallization in the purification of labeled comCH2-CD3) is much greater than that of the phenethyl
pounds, this observation was checked by the National
compound (R = C H ~ - C H Z - C ~ H ~ Ayrey
),
et al. [621
Bureau of Standards, Washington, who confirmed our
also assumed that the Ce-H bond has reached the
results [711. This large isotope effect probably is due to
"half-release" stage in the ethyl compound, and has
the fact that the mutarotation of one or more forms of
progressed beyond this stage in the phenethyl commannose phenylhydrazone is rate-determining for the
pound. However, since our extensive measurements
crystallization.
showed that the ethyl and propyl compounds (R =
CH2-CH2T, CH2-CHT-CH3) have a much smaller
A second example is the distillation of aqueous soluisotope effect than the phenethyl and nitrophenyl comtions of [Tlformaldehyde [421. The reaction involved
pounds( R =CH2-CH2-C6H5,CH2-CHT-C6H4-NO2-g), probably is the hydration and dehydration of the formthe conclusions of Ayrey el aZ.[621 agree with ours
aldehyde. As mentioned in the discussion of secondary
only in that the rupture of the C@-H bond is further
isotope effects, D- and T-labeled molecules react more
advanced for compounds with electron-attracting
sp2 transislowly than unlabeled molecules in sp3
groups than for compounds with alkyl groups.
tions, and correspondingly faster in sp2 -> sp3 transitions. In T-labeled formaldehyde, therefore, for the
In our opinion, the Hofmann degradation shows clearly
equilibria
what is invohed in the determination of isotope effects
and what conclusions can be drawn. Particularly for
hydrogen isotope effects, a definite conclusion can be
drawn only from a large value (cf. however[23al). It is
advantageous to label all points of a molecule at which
CHTOS H20
CHT(OH)2
\ ~T(-H~o)
bonds are broken of formed, and to determine whether
or not isotope effects occur. Generally it is also useful
to study a reaction as a function of the substituents at
the reacting centers.
Thus in T-labeled formaldehyde the equilibrium is disSystematic studies of this type were carried out[691 for the
placed further towards the hydrated form than in the
Dieckmann condensation, the Wolff rearrangement, the
unlabeled compound, so that [Tlformaldehyde is less
hydrolysis of benzyl chloride and para-substituted benzyl
volatile. Equilibria with the dimer, methylene glycol,
or
with oligomeric oxymethylene hydrates may also
[66]E. M . Hodnett and J. J. Flynn jr., J. Amer. chem. SOC. 79,
2300 (1957).
play some part.
--f
AH'')
[67]J . F. Bunnett, Angew. Chem. 74, 731 (1962),Angew. Chem.
internat. Edit. I , 225 (1962).
[68]D. V. Banthorpe, E. D. Hughes, and C. K. Ingold, J. chem.
SOC.(London) 1960, 4054.
[69]A . Fry, Pure appl. Chem. 8, 409 (1964).
926
[70] F. Weygand, H. Simon, and K . D . Keil, Chem. Ber. 92, 1635
(1959).
[71]F. Weygand, H. Simon, K. D . Keil, H. S. Isbell, and L. T.
Sniegoski, Analytic. Chem. 34, 1753 (1962).
Angew. Chem. internat. Edit.
Vol. 5 (1966) / N o . I I
Remarkable isotope effects occur in the paper and column chromatography of T-labeled aldosterones 1721.
The change in the ratio T/14C in intermolecularly
doubly labeled samples was followed. In the paper
chromatography of aldosterone diacetate, for example,
the ratio T/14C for 4 segments of the spot, which were
cut out across the flow direction, were 11.2, 8.5,6.3, and
5.6. Similar fractionations were observed in chromatographic cloumns. It was shown that the fractionation
takes place, not on the basis of the 14C labeling, but
only on the basis of the tritium labeling [*I.
It is not surprising, therefore, that small molecules with
different hydrogen isotope contents, e.g. methanes 1741,
and ethanes [75J, can be completely or largely separated
by normal chromatographic methods. The effects
depend not only on the number of hydrogen isotopes,
but also on their position. Thus Lee and R o d a n d [ 7 6 1
separated position-isomeric T-labeled olefins such as
propylene (CHzT-CH=CHz from CH3-CT=CH2 and
CH3-CH=CHT), cis-butene (CHZT-CH-CH-CH~
from CH3-CT=CH-CH3),
as well as 1-butene
(CH~-CHT-CH=CHZ from CH3-CH2-CH=CHT
and/or CH~-CHZ-CT=CHZ). Traces of tritium in
Table 2.
Isotope effects in chromatographic separations [a].
1
hydrogen gas can be accumulated on an alumina-iron
oxide column at -183 "C r771. Examples of isotope sffects
occurring in column- or gas-chromatographic separations are listed in Table 2.
V. Isotope Effects in Biochemistry
In addition to the equilibrium, kiiietic, and solvent isotope effects defined above other isotope effects can be
observed in biological systems, e.g. changes in Fermeability, inorphologic and genetic changes, changes in
growth rates, and even inability o f the cells to survive 1781. The strongest effects are again produced by
hydrogen isotopes. However, if the s t e p involved in the
process are known, the overall effects can be traced to
primary and secondary isotope effects of labeled substrates in enzyme reactions arid the influence of D20 o n
substrates and enzymes 179-821.
Generally t h e labeled a n d the unlabeled compounds are
present together a n d t h e enzyme-kinetic measurements are
competitive. T h e proportion of t h e labeled compound is
characterized by a t o m weight percentage or by t h e specific
radioactivity. Wi t h deuterium, 100 % isotopic substitution
can b e obtained; in such instances kinetic measurements can
b e carried o u t on each substrate individually. This case,
which is designated a s non-competitive or independent [41, will
b e discussed first.
Chromatography
method
Compound
Isotope effect
(in retention
v01- %, Ibl)
Ion exchange
['4C] Amino acids
-0.123 to 0.338
[TI-2-Aminopurine
[14ClFormic acid
-2.4
0.62 at 6 "C
0.32 at 35.4 "C
[6,7-TzlEstradiol
[ I , 2-TIAldosterone diacetat
0.75
[TIFormic acid
[methyl-TlMethoxyacetic acid
[TlLinoleic acid
[TlCholesterol
acetate
[ I , 2-TIAldosterone diacetate
[ I , 2-TI Aldosterone
2.4
1.01
[77gl
[77gl
0.41
up to 0.49
"731
~721
[77e] E. V. Jensen and H . I . Jacobsen, Recent Progr. Hormone
Res. 18, 387 (1962).
up to 0.73
[77fl
[77f] J. H . Laragh, J. E. Sealy, and P. D. Klein, Proc. Symposium
radiochem. Methods Analysis 2, 353 (1965).
up to 1.63
[77hl
up to -1.15
up to 2.62
[77il
Wjl
Paper
Column
Gas
[TlSteroids
[D]Trimethylsilylatet
carbohydrates
Ref.
[77al
WbI
[77cl
WdI
P7eI
[77fl
0.2
[a1 We are grateful to Dr. P. D . Klein for making a manuscript available
to us before publication.
[bl Retention volume per cent is the percentage difference between the
retention volume at which 50 % of the unlabeled and 50 % of the labeled
compound are eluted. Negative values indicate that the labeled compound flows more rapidly.
[72] P. D . Klein, D . W. Simborg, and P. A. Szczepanik, Pure appl.
Chem. 8, 357 (1964); J. H . Laragh, J. E. Sealei, and P. D . Klein,
in press.
[*] Klein et al. [72] used probit analysis [77k] t o determine
whether the change in the specific activities on purification is due
to isotope effects or to the separation of impurities.
[73] D . S. Sgoutas and F. A . Kummerow, J. Chromatography 16,
448 (1964).
[74] P. L . Cant and K . Yang, J. Amer. chem. SOC.86,5063(1964).
(751 W. A . van Hook and M . E. Kelly, Analytic Chem 37, 508
(1965)
[76] E. K . C . Lee and F. S. Rowland, Analytic. Chem. 36, 2181
(1964).
Angew. Chem. internat. Edit.
1 Vol. 5 (1966) No.
11
[77] J. L . Borowitz and J. R. Gar, Int. J. appl. Radiat. Isotopes
15, 401 (1964).
[77a] K. A. Piez and H . Eagle, Science (Washington) 122, 968
(1955).
[77b] K. A . Piez and H . Eagk, J. Amer. chem. SOC. 78, 5284
(1956).
[77c] H . Gottschling and E. Freese, Nature (London) 196, 829
(1962).
[77d] C . N . Davidson, C . K . Mann, and R . K . Sheline, J. physic.
Chem. 67, 1519 (1963).
[77g] W. G. Brown, L. Kaplan, A. R. Van Dyken, and K . E. Wilzbach, Proc. internat. Conf. peaceful Uses Atomic Energy 15, 16
(1956).
[77h] V. Cejka and E. M . Venneman, Clin. chim. Acta 11, 188
(1965).
[77i] V . Cejka, E. M . Venneman, and P. D . Klein, personal
communication.
[77j] R . Bentley, N. S. Saha, and C. C . Sweeley, Analytic. Chem.
37, 1118 (1965).
[77k] J. D . Finney : Probit Analysis. Cambridge University Press,
London 1952.
1781 J . F. Thomson in : Modern Trends in Physiological Sciences,
Vol. 19. Biological Effects of Deuterium. Pergamon Press, Oxford 1963.
[79] H . Simon, H.-D. Dorrer, and A . Trebst, Z. Naturforsch. 196,
734 (1964).
1801 J. J. Katr, H . L . Crespi, and A . J. Finkel, Pure appl. Chem.
8, 471 (1964).
[80a] J. Katz, R. Rognstad, and R. G. Kemp, J. biol. Chemistry
240, PC 1484 (1965).
[81] V. Moses and M . Calvin, Biochim. biophysica Acta 33, 297
(1959).
[82] H. D . Hoberman, L . Prosky, P. G . Hempstead, and H. W.
Arfin, Biochem. biophysic. Res. Commun. 17, 490 (1964).
927
1. Isotope Effect and Enzyme Mechanism
a) N o n -co m p e t i t ive M e a s ur e m e n t s
vi
_--.- v
Isotope effects of enzymatic reactions with a single substrate can be expressed by the isotope effects of the
maximum rate V and of the Michaelis constant Km.
For the reaction of an enzyme E with a substrate S or a
labeled substrate S', we have, in the simplest caseL831:
From equation (1) we obtain the equations for the
initial rate, vi:
v'~
V'
K,
K,
Further, it follows from equation (6) that in a given
enzyme reaction the isotope effect of vi is a function of
the substrate concentration, and niay be greater or less
than 1.
For two-substrate reactions [including those of the
NAD-dependent dehydrogenases (see Table 3), which
are particularly interesting in connection with D and T
isotope effects], most of the possible mechanisms lead
to the same rate law, which is given by the Dalziel equation (9) [841; this replaces equation (2).
For substrate saturation. we have:
where [Eo] = [El + [ES].
By definition, Km is equal to the substrate concentration at
which vi = V/2. Moreover, it follows from equation (1) that:
(4)
If equilibrium between the enzyme, the substrate, and the
complex ES has been established (k+24 k - l ) , Km is practically equal to the dissociation constant Ks of the complex ES:
[S,] and [Sb] are the concentrations of the substrates or coenzymes. The @ j are the products of the rate constants of the
individual steps; their connection with the mechanism is
given by Dalziel[841, and their determination is also described
in[831. From the CD of an overall reaction (forward and reverse reactions), it is possible, with the aid of the Haldane
equation 1851, the Dalziel equations [841, and equations for
product inhibition [86,871, to derive quantities that characterize a mechanism. The enzyme mechanism can be derived
in a similar and complementary manner from the isotope
effects found for the quantities @.
To illustrate the information obtainable from isotope
effects, consider the liver alcohol dehydrogenase reaction (which was originally assumed to proceed by a
simple Theorell-Chance mechanism [s*l):
(5)
E + CoH
Equation (2) is used to find the constants V, V', K m ,
and Krm, and from these the isotope effects V/V' and
Km/K', are calculated.
In a single-substrate reaction, an isotope effect of V
indicates that an isotope-substituted bond is broken or
formed in the rate-determining step. An isotope effect
of Km can, if condition (5) is satisfied, serve as a measure of the relative affinity of the substrates being compared. For K,/K', > 1,theisotope-substituted substrate
is more firmly bound than the unlabeled substrate.
From equation (2), the isotope effect of the initial rate
vi, which is the quantity that can be measured directly,
is :
For equal substrate concentrations ( [ S ] = [S']), there
follow equation (7) at substrate saturation and equation (8) at low substrate concentrations.
[83] L. Lumper in Hoppe-Seyler Thierfelder: Handbuch der
physiologisch- und pathologisch-chemischen Analyse, VI A,
Enzyme, Part A. Springer, Berlin 1964, p. 17.
928
+ S ; k+Z
' k-1
'
' '
ECoH _____
ECO
\-+SHT
k+3
k-3
E + Co
( C o , CoH, S, and SH represent the coenzyme and the substrate and
their reduced forms)
The parameters of the forward and the reverse reactions are
CD0= l/k+3;@ I = l / k + l ;0 2 = I/k,z;@l,2= k-l/k2k+l
(forward reaction)
(reverse reaction)
For the isotope effects CD/CD'
conditions:
=
'9 we obtain the following
1841 K . Dulziel, Acta chem. scand. 11, 1706 (1957).
[85] R. A. Alberty, J. Amer. chem. SOC.75, 1928 (1953).
[86] R. A. Alberty, J. Amer. chem. SOC.80, 1777 (1958).
[87] W . W. Clelund, Biochem. biophysica Acta 67, 104, 173, 188
(1963).
[88] H. Theorelland B. Chance, Acta chern. scand. 5,1127 (1951).
Angew. Chem. internat. Edit.
1 Vol. 5 (1966) J
No. 11
In Baker‘s investigations [s9] the second and fourth
conditions were not satisfied, so that a simple TheorellChance mechanism probably is not involved. Other
applications of the Dalziel equation are given in [50,89-94].
b) C o m p e t i t i v e M e a s u r e me n t s
the partial rate v, cannot bedetermined independently [*I.
Thus equation (11) gives an isotope effect only for the
ratio VK’,/ V’Km.
If the specific activity of the substrate is [S’]/[S] = No
and that of the product is N p , the isotope effect at low
conversion f131 is
m) [S] a n d [S’] a r e c o m p a r a b l e
The overall rate vt for the transformation of an isotopic
substrate mixture [94d is
v =v
t
S
+- v’
Substitution in equation (11) gives (see also 1971)
S
where v, and v’, are the partial reaction rates of the
labeled and non-labeled substrates, rspectivelq. Since
K,/K’,, may be greater or smaller than 1, each partial
rate may also be greater or smaller than that at the corresponding substrate concentration in the absence of isotope effects. The rate equation (10) corresponds formally to that of a competitive inhibition, but it cannot be
concluded that a labeled substrate acts as a competitive
inhibitor.
Equation (10) shows that the ratio of the partial rates in
the competitive experiment is always proportional to
the ratio of the substrate concentrations 1951.
V/V‘ and Km/K’m can be determined by two methods
in competitive experiments: by determination of vt and
the ratio of the partial rates, or by determination of the
dependence of vt on the ratio of the substrate concentrations [961.
p)
[ S ] i s m u c h g r e a t e r t h a n [S’] ( r a d i o a c t i v e
tracer)
In nearly all experiments with radioactively labeled
substrates, [S] [S] and [S] K,. The information
obtainable from equation (10) is considerably restricted.
vt practically corresponds to the partial rate v,, so that
<<
<
[89] R. H. Baker j r . , Biochemistry 1, 41 (1962).
[90] R. H. Bakerjr. and H. R. Mahler, Biochemistry I , 35(1962).
[91] H . R. Mahler, R. H . Bakerjr., and V. J. Shinerjr., Biochemistry I , 47 (1962).
[92] H. R. Mahler and J. Douglas, J . Amer. chem. SOC.79, 1159
(1957).
1931 J. F. Thomson, J. J . Darling, and L . F. Bordner, Biochem.
biophysica Acta 85, 177 (1964).
[93a] J. F. Thomson and S. L. Nunce, Biochem. biophysica Acta
99, 369 (1965).
[94] J. F. Thomson, D . A . Bray, and J. J . Bummert, Biochem.
Pharmacol. 11, 943 (1962).
[94a] M . B. Thorn, Nature (London) 164, 27 (1949).
[94b] I. A . Rose and S. V. Rieder, J. biol. Chemistry 231, 315
(1958).
[951 R . H . Abeles, W. R. Frisell, and G. G . Mackenzie, J. biol.
Chemistry 235, 853 (1960), erratum in issue No. 6 (1960), no
pagination.
[96] M. B. Thorn, Biochem. J. 49, 602 (1951).
Angew. Chem. internat. Edit.
Vol. 5 (1966) J N o . 11
c) C o m p a r i s o n of N o n - c o m p e t i t i v e a n d
Competitive Experiments
The advantage of the use of highly or completely
labeled substrates is that K , , V, K’m, and V‘, as well
as the kinetic quantities @ and ‘9 (cf. Section V, l a ) can
be determined directly in multi-substrate reactions [50,
89-94]. However, even if the reaction conditions are
closely controlled, the errors in the individual measurements lead to large errors in the isotope effect [5j. This
is shown by a comparison of the results obtained for
alcohol dehydrogenase [50,9*1 and the dependence of the
isotope effect of the urease reaction on various enzyme
preparations 1971.
The competitive procedure has the following advantages: There are no errors due to the comparison of
two sets of measurements, such as are necessary in noncompetitive experiments. In the competitive method,
the isotope effect can be determined with small errors
by radioactive or mass-spectrometric analysis of the
products. Labeling of the required substrates in trace
amounts is generally easier than the preparation of
isotopically pure substrates; e.g. also the preparation of
stereospecifically labeled substrates is unnecessary (cf.
notes to Table 4). The procedure is better suited to in
vivo investigations. One disadvantage is the fact that
tracer labeling generally gives only VK’,,,/ V’K, (cf.
Section V, 1b).
2. Interpretation of the Isotope Effect in Biochemistry
Most publications on enzymatic isotope effects deal with
deuterium isotope effects, and particularly with the study of
hydrogen transfer by oxidoreductases and isomerases. Examples are given in Section V.3 and in Table 3. Although
far less enzyme reactions than organic reactions have been
studied with the aid of isotope effects, some general data o n
the magnitude of isotope effects in enzyme reactions and their
interpretation can be given.
[*] This is most easily seen from the fact that for [S’] 4 K‘m,
equation (2) simplifies to v’i = V‘[S’]/K‘m. Consequently, only
the ratio V ‘ / K m can be determined, and not the individual
quantities.
[97] K . R. Lynn and P . E . Yankwich, Biochem. biophysica Acta
56, 512 (1962); K . R . Lynn and P. E. Yankwich, ibid. 81, 533
(1964).
[98] M . Florkin and E. H . S f o t z in: Comprehensive Biozhemistry,
Vol. 13. 2nd Edit., Elsevier, Amsterdam 1965.
929
Table 3.
Deuterium isotope effects in biochemical redox reactions.
Enzyme and reaction
Substrate
V
V'i
Conditions
and ref.
pH
PI1
2.etc.
V'
-
Alcohol dehydrogenase (yeast)
CH3CHO
NADH
H+ $ CHaCHzOH
+ NAD
CHnCDzOH
rA-4-DI-NADH
1.75
1.75
2.3 1921
1.39
1.31 [h]
Alcohol dehydrogenase (liver)
NADH
H+
CH3CHzOH
CH3CHO
+ NAD
[A-4-D]-NADH
[ B-4-D]-NADH
1.59
0.50 [dl
1.31
1.6 [bl
pH = 8.8; 27OC
r911
1.41
0.81 [dl
0.43
1.59
0.35 [h]
pH = 7.4; 25 "C
[931
1.35
0.69
2.02
0.92 [h]
pH = 7.4; 25 "C
[%I
+
+
+
+
+
Lactate dehydrogenase
Pyruvate
NADH
H+
+
+
+ Lactate + NAD+
Lactate dehydrogenase (ox heart)
NADH
H + + Lactate
Pyruvate
+
+
+ NADt
1.10 [dl
Lactate dehydrogenase (Leuconosfocmesenferoides)
Lactate
NAD+ 7 Pyruvate
NADH
H+
+
+
Malate dehydrogenase
Oxalacetate
NADH
+
+ H+
+
i'- Malate
Glucose-6-P dehydrogenase
NADP+ +
Glucose-6-P
Gluconic acid-6-P
NADPH + H +
+
1.85
1.70
0.82
1.51
1.09 [bl
pH = 7.4; 25 "C
[941
2.0-2.4
1.3-1.6
1.3-1.6
pH = 5.9-9.4
[lo71
not
significant
3.0(16 "C)-2.0(37
'C)
EA(H) = 11700 f 150 cal
ES4(D)= 14935 & 170 cal
[cl
pH = 6.8
11081
1 .o
pH = 6.8; 32°C
[lo81
1.5
pH = 5.9; 27°C
I1081
2.5
[ I 141
+
Formic dehydrogenase (pea)
HCOOH
NAD+ -+ C02
+
+ NADH + H +
Formic dehydrogenase (E. coli)
HCOOH + C02
2 H + 2e-
+
+
Catalase
HCOOH + COz
Hz02
+
1 .o
+ 2 H20
Glycolic oxidase
HOCHzCOOH
NADf +
OHCCOOH
NADH
H+
+
pH = 8.6
[llla]
1.86
+ NAD+
+
+
Succinate dehydrogenase
HOOCCHzCHzCOOH ~2 HOOCCH=CHCOOH
7.45; 24 "C
77% D
0.69
up to 5.9 [991
EACH) = 11250 I 150 cal
EA(D) = 12700 i 300 cal
[cl
~~
Monoamino oxidase
Amine
H20
0 2 -+
+
+
Sarcosine oxidase
Sarcosine -+ HlCO
aldehyde
+ NH3 + H 2 0 2
2.3
+ glycine -+ serine
1.5
0.5
Cytochrome-bs reductase
NADH
H + 2 cytochrome-bs (oxidized)
+ NAD+ i
2 cytochrome-bs (reduced)
[A-4-Dl-NADH
acetyl analogue
pyridinaldehyde
analogue
NADH oxidase
Acceptor: 0 2
NADH-Dehydrogenase
Acceptor: 2,6-Dichloroindophenol and others
[B-4-Dl-NADH
4.5
[B-4-Dl-NADH
1.66--l. 1
+
+
3.66
1.66[1171 10.45
8.7
[a] Isotope effects of the Michaelis constant or of equilibrium constants of the listed substrates in E:. K,/K',
deuterated substrate [cf. eq. (5)l.
pH = 8.1; 25°C
[I001
> 1 indicates firmer bonding of the
[bl Isotope effects for the rate constants of the bonding of coenzymes.
[cl Activation energies (EA) for rupture of the C-H and C-D bonds.
[d] Secondary isotope effects.
The deuterium isotope effects of vi and V in enzyme
reactions are comparable with those of ordinary organic
reactions. Though a value of 6 to 7 is expected for a
normal deuterium isotope effect between 20 and
35 OC[161, the observed effects of, on average 1.5 to 3
still fit into the same scheme if the transition state is
similar to the reactants or to the products. The remarks
in Section 11.1 on the magnitude of the isotope effect as
a function of the loosening of the bond along the reaction coordinate then apply. Moreover, there is often
some compensation and hence reduction of the measurable isotope effect because, in the absence of substrate
saturation, the ratio vi/v'i depends on VK'm/ V'Km
[eqs. (6), (8), and (ll)]. Even when the rate-determining
step is preceded by equilibration, effects of this type
must be expected in the overall isotope effect 141. Consequently, deuterium isotope effects of 6 or more are
seldom attained (see Table 3 and [99, loo]).
The isotope effect of Km is often higher than that expected for equilibrium constants, even when it is known
that K , actually does have the significance of a dissociation constant Ks. For this case M u h l e r [ 9 2 1 and
Shiner [501 believe that there must be interaction between
[99] J . F. Thomson and F. J . Klipfel, Biochem. biophysica Acta
44, 72 (1960).
[loo] P. Sfriftmatter,J. biol. Chemistry 237, 3250 (1962).
Angew. Chem. internat. Edit. 1 Vol. 5 (1966)
No. 11
the enzyme and the isotope-substituted bond already
during binding of the substrate to the enzyme. This
interaction also explains the large isotope effects of K m
found when the substrate contains an isotope-substituted bond that does not take part in the overall reaction, e.g. the 4-B position of NADH for A-specific
enzymes [50,93,94, *I.
An interaction between the substrate and the enzyme
during the binding of the substrate is also indicated by
the hydrogen exchange brought about in dihydroxyacetone phosphate by aldolase even in the absence of an
accept or [94bl.
There are several examples of greater affinity of the
deuterated substrate for the enzyme (KmlK'm > 1)
(see Table 3). Lower affinity of the deuterated substrate
has also been confirmed by stronger inhibition of the
deuterated substrate by a competitive inhibitor [951.
This small isotope effect can be explained by only a partial
rupture of the hydrogen bond in the transition state of the
complex. Such loosening is in agreement with the unexpectedly high isotope effect of the substrate dissociation constants, which is explained by an interaction between the
enzyme and the C-H bond involved in the hydrogen transfer.
According to Silverstein and Boyer[lo3], the cleavage of the
N A D or N A D H product is the rate-determining step. In
this case the above assignment of the isotope effect to the
kinetic constants would be invalid. Miiller-Hill and Wallenfels [lo41 concluded from the temperature dependence of the
reaction that the first of these mechanisms applies below
20 "C, and the second above 20 "C.
We have studied the temperature dependence of the
isotope effect of [I-Tlethanol and [A-4-T]NADH as
substrates of alcohol dehydrogenase (from yeast) [*05,
105al. The increase in the isotope effect with rising
temperature (Table 4) can be explained by a change in
the rate-determining step.
While the isotope effect of Km can provide information about
the binding of the substrate, and hence about the active site
of the enzyme, the isotope effect per se can be used as evidence
of changes in the active site. A good example is found in
native and carboxypeptidase-treated aldolase "011. While the
native enzyme showed no isotope effect in the synthesis with
labeled triose phosphates, a deuterium isotope effect of 6 and
a tritium isotope effect of 20 were found for the treated
enzyme "011.
Since alcohol dehydrogenase of yeast also catalyses the
oxidation of the homologues of ethanol at comparable
rates, we were able to use this enzyme to study the influence of various substrates on the isotope effect in an
enzyme reaction [lo51 (see Table 4). The increase in the
isotope effect from ethanol to propanol is much greater
than the accompanying change in the reaction rate,
which is only 34% [1061.
3. Specific Examples
Table 4. Comparison of the isotope effects ( k H / k T ) in the reaction of
some alcohols with alcohol dehydrogenase (yeast) under various conditions [105].
The examples described here are arranged in accordance with
the enzyme list [981. The importance of the isotope effect for
the elucidation of enzyme mechanisms can be shown inter
alia for the participation of a n enzyme in a coupled system,
stereospecificity of a Caabd molecule, intramolecular proton
shift, and cleavage of a molecule into identical fragments.
a) H y d r o g e n I s o t o p e E f f e c t
Measurements of the isotope effect in the alcohol dehydrogenase (yeast) reaction with [4-D]-NADH (chemically and enzymatically reduced) and [l,l-D2]-ethanol
have been reported [50,91,92,1021. The authors assume
that the rate-determining step is the transformation of
the ternary complex
ECoSH
A ECoHS
\ k-
the isotope effect of which is k,/k',
(Table 3).
=
k-/k'-
=
1.75
[*I NAD+ (NADP+)= nicotinamide adenine nucleotide (phosphate); NAD (NADPH) = reduced forms of NAD+ and NADP+;
[A-4-D]NADH: in the nicotinamide portion of NADH, the
4-position facing forward when the atoms are numbered 1 to 6
in the anticlockwise direction is now labeled, i.e. [4R-4-D]NADH [100a]. Similarly, [B-4-D]NADH is [4S-4-D]NADH.
Depending on the stereospecificity of the hydrogen transfer,
enzymes that transfer hydrogen to the A position of NADH are
known as A-enzymes, and substrates of the type CHHbd are
divided into A- and B-substrates.
[100a] J. W. Cornforth, G . Ryback, G . Popjak, C. Donninger, and
G . Schroepfer jr., Biochem. biophysic. Res. Commun. 9, 371
(1962).
[loll I . A . Rose, E. L . O'Connell, and A . H. Mehler, J. biol.
Chemistry 240, 1758 (1965).
[lo21 H. R. Levy, F. A . Loewus, and B. Vennesland, J. Amer.
chem. SOC.79, 2949 (1957).
Angew. Chem. internat. Edit.
1 Val. 5 (1966) 1 No. 11
Conditions
I5 "C
pH 7.6
25 "C
[I-TlEthanol [a1
1
4.1 I 0.05
[I-TlPropanol [a]
I
6.7 i 0.2
[I-TlButanol [a]
1
6.8 L 0.2
[a] Mixture of the two stereoisomers. With tracer amounts of the tritiated
substance, there is no appreciable double labeling, so that there is no need
to use stereospecifically labeled substrates when the isotope effect is
measured on the [A-4-T]-NADH formed.
For the alcohol dehydrogenase of liver [911, an iterative
method was used to find the 1 3 kinetic constants from the 12
independent equations obtained for the forward and reverse
reactions. Isotope effects could then be calculated for each
step of a complex mechanism. In addition to the values in
Table 3, k H / k D = 2.28 was obtained for the hydrogen
transfer from CH3CH20H to NADf, and k H / k D = 3.11 for
the transfer from [A-4-D]NADH to CH3CHO.
In the determination of the isotope effect of [A-4-T]NADH in the alcohol dehydrogenase reaction with
acetaldehyde, propionaldehyde, or butyraldehyde, a
substrate dependence is observed with the enzyme from
yeast [105al, but not with that from liver [105bl. This
confirms that the rate-determining step in the reaction
of the liver enzyme depends on the dissociation of the
enzyme.
[lo31 E. Silverstein and P. D . Boyer, J. biol. Chemistry 239, 3908
(1964).
[lo41 B. Miiller-Hill and K . Wallenfels, Biochem. Z . 339, 338
(1964).
11051 D . Palm, Z. Naturforsch. Zlb, 540 (1966).
[105a] D . Palm, Z . Naturforsch. 2lb, 547 (1966).
[105b] D . Palm and T.Fiedler, unpublished.
[lo61 J . van Eys and N . 0. Kaplan, J. Amer. chem. SOC.79, 2782
(1957).
93 1
Thornson [92,9301 studied isotopeeffects of [A-4-D]NADH
and [B-4-D]NADH in the lactate dehydrogenase reaction. [B-4-D]NADH gives an inverse isotope effect in
relation to the normal isotope effect of V due to
[A-4-D]NADH (Table 3); this points at least to the
interaction of both hydrogens in position 4 of the
NADH with the enzyme in the course of the reaction.
From the isotope effect of the NADP-dependent glucose
6-phosphate dehydrogenase, it was possible to determine the rate-determining step for the formation of CO2
from glucose in whole cells. This is a C-H cleavage,
either during the H transfer from glucose to NADP or
during the reoxidation of the NADP. The Same was
found for tumor cells 11071.
Whereas various substrates have been studied with the
one enzyme alcohol dehydrogenase, Aebi 11081has carried
out comparative measurements with one substrate
(formate) and various enzymes (Table 3).
The succinate dehydrogenase reaction on deuterated
succinic acid is a classic example of enzymatic isotope
effects 11091. With perdeuterated substrates, it was later
found that V/V‘= 5.9 1993. However, secondary isotope
effects must also be taken into account in the case of the
[D4]succinate. As a substrate of the respiratory chain,
[D4]succinate has no retarding effect on the oxidative
phosphorylation.
The elucidation of a stereospecific reaction was achieved
by Belleau [11OJ for the enzymatic decarboxylation of
amino acids to aniines and the dehydrogenation of the
latter. The two enantiomeric [I-Dltyramines were prepared, and their isotope effects in the monoaminooxidase reaction with the decarboxylation product obtained from tyrosine in D20 were compared.
An isotope effect was expected in the dehydration of
malate (fumarase reaction), but none was found 11111.
Since the rupture of the stereospecifically labeled
carbon-hydrogen bond is not rate-determining, one
must postulate a mechanism involving a malate-X
complex that is as yet unknown.
malate+ E
$3malate-E
+
malate-X
E-fumarate
An isotope effect of 2 was found for deuterium
(about 3.2 for tritium) in both directions in the isomerase reaction. A similar isotopt. effect was found [1131 for
the triose phosphate isomerase. [3-D]1,3-Dihydroxyacetone-1 -phosphate reacts 2.2 times more slowly than
the unsubstituted substrate.
In the epimerization of uridine diphosphate galactose
and gluccse by an enzyme from yeast, an inverse isotope effect of ICHIkT =- 0.66 to 0.33 was found for the
[4-T]labeled hexoses 1113al. The direction and magnitude of this isotope effect are in agreement with a
mechanism in which the first step is a reversible hydrogen transfer from C-4 to the enzyme. Inverse isotope
effects also have been found for an equilibrium of this
type in the case of alcohol dehydrogenase (Table 3).
This indicates a rearrangement of the enzyme coniplex, the hydrogen taking up the appropriate position
for transfer to the epimeric position of the hexose.
From the magnitude of the equilibrium isotope effect
it is concluded that C, N, or 0 atoms are involved in the
hydrogen transfer, and the participation of S can be
excluded.
An interesting application of deuterium and tritium
isotope effects was demonstrated [114,1151 in the elucidation of various steps in the ribulose diphosphate carbI-
HzCOP
1
14C=O
I
HCOH
’
xp
HzCOP
-1
I
H014C-COOcI =o
I
1-
HCOH
HICOP
HzCOP
~~0
A
DldOH
HzCOP
+
I
COOH
1
I
HCOH
I
COOH
(5a)
“c-2 cleavage”
s
+
E + fumarate
In the racemization of [2-D]lactate by an extract from
Clostridium butyiicum, an isotope effect of H k / k D = 2.14
was observed for both enantiomers. The fact that no
deuterium was lost as a result of the racemization was
shown by the fact that the rate of dehydrogenation by
the lactate dehydrogenase both for the D- and for the
L-form corresponded exactly to that of the deuterated
lactic acids [ I l l a J .
[lo71 I. A. Rose, J. biol. Chemistry 236, 603 (1961).
[lo81 H. Aebi, Pure appl. Chem. 8, 459 (1964).
[lo91 H . Erlennzeyer, W . Schoenauer, and H. Siillman, Helv.
chim. Acta 19, 1376 (1936).
[ l l O ] B. Belleau and J. Burba, J. Amer. chem. SOC. 82, 5751
(1960); B. Belleau, M . Fang, J . Burba, and J. Motan, ibid. 82,
5752 (1960); B. Belleau and J . Moran, Ann. New York Acad.
Sci. 107, 822 (1963).
[ l l l ] R. A . Alberty in P . D. Boyer, H . Lardy, and K . Myrback:
The Enzymes, Vol. 5. Academic Press, New York 1961, p. 531.
[ l l l a ] S. S. Shnpiro and D . Dennis, Biochemistry 4, 2283 (1965).
932
Measurement of the deuterium isotope effect in the
hexose isomerase reaction of [1-14C-2-D]glucose 6phosphate showed the occurrence of an intramolecular
proton transfer 11121. In the substrate-enzyme complex,
about 50% of the hydrogen is transferred as protons
from C-2 into position 1of the substrate (and vice vcrsa)
without exchange with the solvent.
I
1-
HOLOOHZCOP
-1
I
H ~ O H
DCOH
I
I
COOH
COOH
“c-4 cleavage”
[112] I. A . Rose and E. L. O’Connell, J. biol. Chemistry 236,
3086 (1961).
11131 S. V. Rieder and I. A . Rose, 3. biol. Chemistry 234, 1007
(1959).
[113a] R . D. Bevill I l l , E. A . Hill, F. Smith, and S. Kirkwood,
Canad. J. Chem. 43, 1575 (1965).
[114] G . MiilZhofer and I. A. Rose, J. biol. Chemistry 240, 1341
(1965).
[115] I. A. Rose, G . Miillhofer, and F. Fiedler, unpublished.
[116] B. Mackler, Biochem. biophysic. Res. Commun. 4, 195
(1961).
[117] P. Strittmatter, J. biol. Chemistry 239, 3043 (1964).
[118] J. L. Rabinowitz, T. Sall, J. N . Bierlyjr., and 0 . Oleksyshyn,
Arch. Biochem. Biophysics 63, 437 (1956).
[119] J. L . Rabinowitr, J. S . Lafair, H . D . Strauss, and H . C.
Allen j r . , Biochem. biophysica Acta 27, 544 (1958).
[120] S . Seltzer, G. A . Hamilton, and F. H. Westheimer, J. Amer.
chem. SOC.81, 4018 (1959).
Angew. Chem. internat. Edit. 1 Vol. 5 (1966)
1 No.
11
oxylase reaction (carboxydismurase). The object was to
find whether the C02 is attached to C-2 or C-4 of the
ri bulose diphosphate ( 4 ) before cleavage into two molecules of 3-phosphoglyceric acid ( 5 ) . An intramolecularly doubly labeled [2-14C-2-D]phosphoglyceric acid
(5a) can be formed as well as the unlabeled acid only if
the C02 is attached to C-2. Since the [14C]glycolic acid
obtained from the phosphoglyceric acid exhibited an
isotope effect of 2.5 on oxidation with glycolic acid
oxidase, this glycolic acid must have contained D as
well as 14C. i.e. the CO;? is attached to C-2 in (4). 3Tritiated (4) reacts four to six times more slowly than
the H compound in the carboxylation at pH 8.1 and
25 "C. The release of the proton appears to be the limiting step of the overall reaction 11151.
In the irreversible formation of a C-H bond by proton
abstraction from a solvent in which the hydrogen is
partially labeled, a much smaller content of the higher
hydrogen isotopes is usually found in the product than
in the solvent. This is a result of the isotope effect in the
cleavage of an X-H bond. where X may be 0, S, N, etc.
A typical example, which has been studied both experimentally and theoretically, is the hydration of isobutylene
in acidic media 11211. When this reaction is carried out in
I-I20/D20 mixtures, the concentration of D in the
t-butyl alcohol formed is 3.9 times lower than that in the
water; the ratio for T is 7.1, and that for T in D20 is 1.9.
Corresponding biochemical examples are : In the enzymatic
cleavage of citrate in HOT, T is incorporated into acetyl
CoA. Hydrogen is incorporated preferentially, in the ratio
kH/kT = 6.7 [1221. In the cleavage of threo-D,-isocitrate to
[121] V. Gold and M. A . Kessick, J. chem. SOC.(London) 1965,
6718 (1965).
11221 J. B o w , R. 0. Martin, L. L. Ingraham, and P. K. Stumpf,
J . biol. Chemistry 234, 999 (1959).
succinate and glyoxylate, an isotope effect of kH/kT = 6.5 to
13.9 is found, depending on the isocitrate concentration
(2x 10-3 to 4x 10-3 M ) , when the reaction is carried out in a
buffer solution containing tritium [1231. In the cleavage of
phosphoenol pyruvate by pyruvate kinase in HOT, T is incorporated with an isotope effect kH/kT of 6.4 [Iz4].
b) I s o t o p e E f f e c t s of C a r b o n
Lynn and Yunkwich[971studiedthe isotope effect of the urease
reaction (with urea having the natural 12C/13C ratio) as a
function of conversion, concentration, buffer, temperature,
pH, and aging of the enzyme, and found a maximum value
of 2.3%. The differences of the isotope effects found for
various enzyme preparations were in some cases larger than
the differences found for the above variables. The isotope
effect of 5 to 10% found by other authors could not be
confirmed.
A 14C isotope effect of an unusual magnitude was found[119]
for the enzymatic oxidation of [14C]formate by formic acid
dehydrogenase. The value of 1.27 at 37 "C was independent
of the substrate activity, which varied between 0.8 and 100
pCi/mmole.
The decarboxylation of oxalacetate to pyruvate by a n enzyme
from Micrococcus lysodeikticus shows no isotope effect with
the I3C-labeled substrate. The Mn(n)-catalysed, non-enzymatic decarboxylation, on the other hand, gives a n isotope
effect of 1.06; solvent isotope effects in D20 indicate that the
limiting step is not the actual decarboxylation [1*01.
Received: August Sth, 1965; in final form July 22nd, 1966 [ A 544IE1
German version: Angew. Chem. 78, 933 (1966)
Translated by Express Translation Service, London
--
[123] H . H . Daron, W. J. Rutter, and I . C . Gunsalus, Biochernistry 5, 895 (1966).
[124] H . Simon and G. Miillhufer, unpublished.
Progress in the Chemistry of Allene
BY DR. K. GRIESBAUM
CENTRAL BASIC RESEARCH LABORATORY, ESSO RESEARCH AND ENGINEERING
COMPANY, LINDEN, NEW JERSEY (U.S.A.)
Considerable progress has been made in the past decade in the preparation and chemistry of
allene. The present article is a critical review of this progress, and shows that owing to tho
particular structure of allene (two double bonds situated in mutually perpendicular planes),
the chemistry of this compound is unique in many respects.
I. Introduction
The chemistry of acetylene and of butadiene, i.e. the
smallest hydrocarbons containing a triple bond and two
conjugated
bonds
has been
known for a number of years, and both compounds are
used as raw materials for huge industries [I]. on the
other hand allene, i.e. the parent compound of the
Angew. Chem. internat. Edit.
/ Vol. 5 (1966) J No. 1I
cumulenes, has found hardly any industrial use, and its
chemistry, unlike that of acetylene and of butadiene,
remained underdeveloped for a long time. Thus even a
few years ago, little was known about such fundamental
~~
[l] See, e.g., W. Reppe: N e w Entwicklungen auf dern Gebiet
der Chernie des Acetylens und Kohlenoxids. Springer, Heidelberg 1949; Y. Mayor, Ind. Petrole 32, 62 (1964); 1. KirshenbaNm
and R. P. Cahn in: Encyclopedia of Chemical Technology. Interscience, New York 1964, VOI. 111, p. 784.
933
Документ
Категория
Без категории
Просмотров
1
Размер файла
1 439 Кб
Теги
chemistry, effect, organiz, biochemistry, isotopes
1/--страниц
Пожаловаться на содержимое документа