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Study of the Mechanism of Dehydrogenase Reactions by Measurement of the Isotope Effects.

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The relevant publications from the Hahn-Meitner-lnstitut,
numbering fourty up to the present, were reviewed.
[VB 121 IE]
Lecture at Darmstadt (Germany) on May 28, 1968
German version: Angew. Chem. 80, 706 (1968)
[*] Prof. Dr. K. E. Zimen
Hahn-Meitner-Institut fur Kernforschung
1 Berlin 39, Glienicker Str. 100 (Germany)
Study of the Mechanism of Dehydrogenase
Reactions by Measurement of the Isotope Effects
By D . Palm [*I
Classical kinetic methods for the study of enzyme reactions
have recently been extended by methods for selective determination of fast or slow steps in complex reaction sequences.
Slow and irreversible steps can be detected also in enzyme
kinetics by differences in the reaction rate of isotopically
labeled substrates. This is particularly so for the isotope
effects (IE’s) of the hydrogen isotopes, for which primary and
secondary IE’s can be detected 111. Thus hydrogen transfer in
NAD-dependent dehydrogenases is particularly suited for
investigation of the empirical and theoretical relation of IE
to enzyme-kinetic values.
The primary IE (4.1 to 6.8 at 25OC) for the substrates
[I-TI-ethanol, -1-propanol, and -1-butanol confirm the required rate-determining hydrogen transfer for alcohol dehydrogenase (ADH) from yeast, but an increase in IE with
rising temperature also shows a change in the rate-determining step. For the ADH of liver the small secondary IE
(1.4 to 1.6 at 25 “C) for the homologous alcohols is in agreement with a rate-determining dissociation of the enzyme
NADH complex, which is independent of the substrate.
The reverse reaction, studied with the stereospecifically
labeled [A-4-T]NADH again shows primary IE’s of 2 to 5
(depending on the corresponding homologous aldehydes) for
yeast enzyme, whereas only a small difference from unity was
found for the liver enzyme. The sterically hindered substrate
2-methylcyclohexanone for Iiver ADH stands out with an
IE of 3.6; since 2-[methyZ-T]methylcyclohexanone reacts
4.5 % faster, the site of steric hindrance can be more accurately localized [ZJ.
In the case of lactate dehydrogenase of rabbit muscle the IE
of 2.5 for ~-[ZT]lactatecorresponds to a product of two
secondary IE‘s, which are due to isomerization and dissociation of the enzyme-NADH complex. This interpretation
excludes a kinetic influence of ternary complexes. The reverse
reaction with [A-4-T]NADH also shows an IE of up to 2.0
that indicates isomerizations, but under the influence of
pyruvate inhibition at concentrations > 1 x l o - 3 ~the IE
disappears.
Finally, a mechanism similar to that for yeast ADH can be
ascribed to glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.
[VB 161 IE]
Lecture at Konstanz (Germany), on May 30. 1968
German version: Angew. Chem. 80, 706 (1968)
[*I Doz. Dr. D. Palm
Chemisches Institut Weihenstephan und
Organisch-Chemisches Institut der Technischen Hochschule
8 Munchen, Arcisstr. 21 (Germany)
[l] H. Simon and D . Palm, Angew. Chem. 78,993 (1966); Angew.
Chem. internat. Edit. 5, 920 (1966).
121 D. Palm, Z. Naturforsch. 216, 540, 547 (1966); D. Palm,
T. Fiedler, and D . Ruhrseitz, ibid. 236, 623 (1968).
Oxidation of Pyrrolic Impurities in Polyamides
By P. Schlack[*l
When prepared under not quite perfect conditions, polyamides may contain pyrrole groups formed in side reactions
and detectable by Ehrlich’s reagent. In nylon 6 (polycapro-
740
lactam) pyrroles are observed particularly if 1,Il-diaminoundecanone is formed by loss of water and COz from two
moles of 6-aminohexanoic acid or if the corresponding anhydro base is formed from c-caprolactam by loss of water.
This ketimine is very sensitive to oxygen and after autoxidation gives an intense pyrrole reaction.
Polycaprolactam fibers (“Perlon”) that contain units of this
imine show a strong tendency to yellow on fairly long exposure to light in the presence of oxygen or on thermal oxidation. Rochas and Martin have found that polyamide fibers
(nylon 66) may also give a positive pyrrole reaction after
photoxidation 111. They assumed that the pyrroles were formed by reaction of amino end groups in the fiber with a,cr’-dioxoadipic acid formed by oxidation of adipyl groups. Mar&
and Larch came to the conclusion that it was almost wholly
the diamine groups that were involved in pyrrole formation;
they held that primary attack by the oxygen was always o n
the methylene groups next to the amide nitrogen 121.
In studies of the yellowing of polyamide textiles in our Institute, F. Sommermann [31 found that pyrroles are also formed
on oxidation of N-free olefinic substances such as 3-heptene,
oleic acid, methyl linoleate, and squalene in the presence of
prjmary amines or amino acids and also on subsequent addition of such amines. Since sweat contains glycerides of polyunsaturated fatty acids, squalene, ammonium salts, and other
nitrogenous bases, pyrroles or their colored oxidation products could be formed on textile substrates even without
chemical participation of the fiber substance.
Only the primary amino end groups of the fiber come into
question as a nitrogen source for pyrrole formation within
the fiber substance. If however, the amino groups are inactivated by acylation, e.g. by acetic anhydride [4J, then pyrrole
can no longer be formed on the fiber unless ammonia or
amino groups are newly formed. Autoxidation in light is then
also greatly hindered. Most of the amino groups can be
blocked under remarkably mild conditions that can be realized in practice, nameIy, by impregnating the textile goods
with solutions of anhydride-forming aromatic polycarboxylic
acids, e.g. trimellitic acid, then drying them, and heating
them for one to two minutes at 170-180°C by passage
through a thermofixing apparatus. The tendency to yellowing
is much reduced by this pretreatment.
From the UV spectra of the dyes formed in the fiber by p (dimethy1amino)benzaldehyde it can be concluded that both
a- and P-methine dyes occur side by side, whereas the dyes
formed from unsaturated fatty acid esters and amino acids
appear to consist mainly of @-derivatives.
[VB 164 IE]
Lecture at Karlsruhe (Germany) on May 30, 1968
German version: Angew. Chem. 80, 761 (1968)
___
[*I Prof. Dr. P. Schlack
Deutsche Forschungsinstitute fur Textilindustrie
Reutlingen-Stuttgart
Institut fur Chemiefasern
7 Stuttgart-Wangen, Ulmer Strasse 227 (Germany)
(11 P. Rochas and J . C. Martin, Bull. Inst. Textile France 83, 41
(1959).
[2] B. Marek and E. Lerch, J. SOC. Dyers Colourists 81, 481
(1965).
[3] F. Sommermann, unpublished.
[4] F. H . Steiger, Textile Res. J. 27, 459 (1957); see also [I].
Aromatic Sigmatropic Rearrangements
By Hans Schmid [ *I
Aromatic sigmatropic rearrangements are thermal reactions
whose transition states can, to a first approximation, be
considered as interaction complexes between two pseudoradical halves that have arisen by homolysis of the bond that
is attacked. At least one of these halves must be aromatic in
nature, i.e. its x-system is to be described by aromatic molecular orbitals. A well-known example is the thermal Claisen
rearrangement of ally1 aryl ethers that occurs with inversion
Angew. Chem. internat. Edit.
Vol. 7 (1968) 1 No. 9
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