close

Вход

Забыли?

вход по аккаунту

?

Lysozymes A Chapter of Molecular Biology.

код для вставкиСкачать
ANGEWANDTE CHEMIE
VOLUME 8 . N U M B E R 4
APRIL 1 9 6 9
PAGES 2 2 7 - 2 9 4
Lysozymes: A Chapter of Molecular Biology
By P. Jolltsr*l
Hen egg-white lysozyme was the first enzyme whose tertiary structure could be elucidated.
The peptide chain of this enzyme is arranged in two seciions, of approximately equal size,
that are separated by a deep cleft. Substrates (and inhibitors) are bound in this cleft via
hydrogen bonds and are hydrolyzed under the action of Glu 35 and Asp 52, which form
the active site of the enzyme. Although the lysozymes, which occur in many species of
animals and plants, exhibit differences in their chemical behavior, they have the same
qualitative biological activity; quantitatively important differenceshave been noted which
also concern the specificity. Infection of E. coli with bacteriophages gives rise to a
lysozyrne whose formation is controlled by the phage DNA. The fact that mutated lysozymes are produced when the phages are treated with mutagens opens new fields of research in molecular biology.
1. Introduction
Hen egg-white lysozyme (EC 3.2.1.17) is one of the
most extensively studied enzymes: its primary structure, the disposition of its disulfide bonds, its spatial
behavior (first three-dimensional structure of an
enzyme molecule), its active center, specificity, and
mode of action have all been investigated in detail.
Lysozymes are not only found in hen and other bird
egg-whites but are widespread enzymes, occurring in
many human tissues and secretions, and in various
other vertebrates and invertebrates; their presence has
also been established in bacteria, phages, and plants.
For these reasons, many fruitful comparative studies
have been carried out, concerning mainly the relationship between their chemical structure and their
biological activity. The purpose of the present review
is not to summarize the research done since 1963 [I],
but rather to draw the attention of the reader to
some particular points and developments that are of
interest for the study not only of the lysozymes but
also of other enzymes.
[*I
Prof. Dr. P. Jo1lt.s
Laboratoire de Chimie biologique, Faculte des Sciences
de I’Universite
96, Boulevard Raspail, Paris V l e (France)
[I] P. JollPs, Angew. Chern. 76, 20 (1964); Angew. Chem. internat. Edit. 3, 28 (1964).
Angew. Chem. internat. Edit. 1 Vol. 8 (1969) I No. 4
2. Hen Egg-White Lysozyme
2.1. Primary Structure and Disulfide Bonds
The sequence of hen egg-white lysozyme was determined by Joll2s et al. [2+31 and by CanJield[41;it is a
protein containing 129 amino-acid residues which
Fig. 1. Primary structure and disulfide bonds of hen egg-white lysozyme.
r2] J. Jolles and P. Joll2s, C. R. hebd. Seances Acad. Sci. 253,
2773 (1961).
[3] J . JoUks, J. Jauregui-Adell, I. Bernier, and P . Jolles, Biochim.
biophysica Acta 78, 668 (1963).
[4] R. E. Canfield, J. biol. Chemistry 238, 2698 (1963).
227
form a single polypeptide chain cross-linked at four
places by disulfide (-S-S-) bonds c5-71 (Fig. 1).
quite close to the 3.010 type of hydrogen bond arrangement and an isolated turn (119 to 122) of 3.010 helix
(Figure 2).
2.2. Three-Dimensional Structure
The conformation of lysozyme has been determined
by X-ray analysis@]. The method depends on the
preparation and study of a series of protein crystals
into which additional heavy atoms, such as uranium
atoms, have been introduced without otherwise affecting the crystal structure. The first successful applications of this method were in the study of spermwhale myoglobin by Kendrew and of horse hemoglobin by Perutz. Comparison of the electron density
distribution at 2 A resolution with the primary structure revealed the spatial arrangement of each of the
129 amino acid residues of hen egg-white lysozyme
and permitted the positions of some 95 % of the atoms
to be located to within about 0.3 A.
The molecule may be considered in two parts. The
first 40 residues form a right-hand wing in which the
peptide chain is coiled twice around a core of nonpolar residues in a stable-looking conformation (crhelix structure). Residues 41 -95 branch out to form a
left-hand wing which appears to have a less rigid conformation and contains a high proportion of polar
residues. The remainder of the chain partially closes
the gap between the two wings and wraps around the
outside of the right-hand wing leaving a pronounced
cleft between the two parts of the molecule @I.
In contrast to the myoglobin molecule, which consists of long stretches of cr-helix linked by generally
short non-helical corners, the a-helix content of lysozyme is only about 40%; there is evidence that nearly
all of the helices are distorted to some extent from the
standard structure, usually without affecting the
number of residues per turn of helix[lol. There are
three runs of helix, each about 10 residues long (5 to
15, 24 to 34, 88 to 96) for which the axial translation
per residue and number of residues per turn fall close
to the cr-helix values of 1.5 A and 3.6, respectively,
given by Pauling and Covey (Table 1).There is a further
shorter length of helix (80 to 85) which appears to be
Table 1. Parameters of helical regions of hen
egg-white lysozyme [lo].
per turn
5-15
24-34
80-85
88-96
1.51
1.485
1.66
1.455
100.2
98.3
106.6
96.7
3.59
3.66
3.38
3.72
I51 P. .loll&, J . Jauregui-Adell, and J . Joll&, C. R. hebd. Seances
Acad. Sci. 258, 3926 (1964).
[6] J. R . Brown, Biochem. J. 92, 13 P (1964).
[7] R . E. Canfield and A . K . Liu, J. biol. Chemistry 240, 1997
(1965).
[8] C. C. F. Blake, D . F. Koenig, G . A . Mair, A . C. T . North,
D . C. Phillips, and V. R. Sarma, Nature (London) 206,757 (1965).
[9] L. N . Johnson, Exposes annu. Biochim. med. 27, 31 (1966).
[lo] C. C. F. Blake, G . A Mair, A C. T . North, D . C. Phillips, and
V. R. Sarma, Proc. Roy. SOC.(London), Ser. B 167, 365 (1967).
228
Fig. 2. A single turn of 3.010helix, showing also the conformation of
Val 120 1101. The hydrogen bonds are represented by dashed lines.
Fig. 3. Region between Ala 42 and Gly 54 where the main chain folds
back on itself to form an antiparallel pleated sheet arrangement [lo].
The hydrogen bonds are represented by dashed lines.
The acidic (Asp and Glu) and basic (Lys, Arg, and
His) side chains and the terminal groups are all distributed over the surface of the molecule; this is also true
of the remaining polar side chains, with two exceptions
(Glu 57; Ser 91). In contrast, the great majority of the
non-polar or hydrophobic side chains are in the interior of the molecule [lo], where they are by no means
distributed uniformly throughout the length of the
chain. There is none between residues 38 and 55 and
the deep cleft in the molecule seems to divide it into
two parts of markedly different character (right-hand
and left-hand wings, see above). In fact, lysozyme,
like myoglobin, is quite well described as an oil drop
with a polar coat [131.
[Ill L . Pauling and R . B. Corey, Proc. nat. Acad. Sci. USA 37,
729 (1951).
[I21 R. E. Marsh, R . B. Corey, and L . Pauling, Acta crystallogr.
8, 62 (1955).
[I31 D . C. PhiNips, Sci. American 215, 78 (1966).
Angew. Chem. internat. Edit.1 Vol. 8 (1969) / No. 4
2.3. The Activity of Lysozyme
FH20H
Simultaneously with his discovery of lysozyme,
Neming 1141 discovered a gram-positive species of
bacteria, Micrococcus lysodeikticus, which is particularly susceptible to the action of the enzyme. It was
many years later when Salton [I51 demonstrated that
the substrate is located entirely within the bacterial
cell wall. An indication of the type of linkage attacked
by lysozyme was first established when Berger and
Weiser “61 showed that lysozyme also degrades chitin,
the linear polymer of N-acetylglucosamine (NAG).
They concluded that the enzyme possesses p (1 +4)
glucosaminidase activity.
CHzOH
HO OH
H o D H , O H
NHAc
/O
NHAc
Salton and Gltuysen [I71 subsequently isolated a tetrasaccharide containing equimolar amounts of NAG and Nacetylmuramic acid @AM) from lysozyme digests of M .
lysodeikticus cell walls. On incubation with lysozyme, this
tetrasaccharide yielded a disaccharide containing NAG and
NAM. It was deduced that the tetrasaccharide is a dimer
of this disaccharide in which the two units are joined by the
lysozyme-sensitive @ (1 +4) linkage. Its complete structure
was established only in 1963 by Jeanloz et al.[lsl as Nacetylglucosaminyl-P-(1 +4)-N-acetylmuraminyl-P-(l +4)-Nacetylglucosaminyl-P-(1 +4)-N-acetylmuramic acid ( 1 ) .
CH~OH
NHAc
CHzOH
(6)
NHAc
oo;o
Lysozyme
CH3CgCOOH
(7)
H,OH
CH3CkCOOH
CH~C~COOH
NAG
NAM
NAG
NAM
(1 )
The proposed structure of the cell-wall mucopolysaccharide, containing alternate units of NAG and
NAM residues joined by p (1 +4) linkages, is very
similar to the structure of chitin, the only difference
being the inclusion of NAM residues in the former;
it is therefore probable that the effects of lysozyme
upon chitin and upon the bacterial cell walls are
very closely related. In fact, a large proportion of the
recent work on the activity of lysozyme has been concerned with its properties as a chitinase, especially
during its interaction with relatively short oligomers of
NAG. NAG (2) and other simple amino-sugar molecules (3)-(7), including trimeric NAG (8), act as
competitive inhibitors of the activity of lysozyme by
binding to the enzyme in the same way as a part of the
large substrate molecule. Experiments at low resolution with low molecular weight inhibitors related either
t o the bacterial cell wall substrate or to the poly-NAG
substrate have shown that the sugars bind the lyso1141 A. Fleming, Proc. Roy. SOC.(London), Ser. B 93, 306 (1922).
[15] M . R . J . Salton, Nature (London) 170, 746 (1952).
[16] L. R. Berger and R . S . Weiser, Biochim. biophysica Acta 26,
517 (1957).
[171 M . R. J. Salton and J . M . Ghuysen, Biochim. biophysica
Acta 36, 552 (1959); 45, 355 (1960).
[181 R . W. Jeunlor, N. Sharon, and H . M. Flowers, Biochem. biophys. Res. Commun. 13,20 (1963).
Angew. Chem. internat. Edit.
1 Vol. 8 (1969) 1 No. 4
HO OH
NHAc
CHzOH
NHAc
(8)
zyme at a number of different places in the cleft between the right-hand and left-hand wings of the lysozyme molecule [9,19,201.
Trimeric NAG (8) fills the top half of the cleft and is
bound to the enzyme by a number of interactions; six
strong hydrogen bonds have been identified, two of
them connecting an NAG residue to the side chains of
the tryptophan residues 62 and 63. Among the more
important nonpolar interactions are those between the
NAM residue and the ring system of Trp 62: these deserve special mention because they are affected by
small changes in the conformation of the enzyme
molecule that occur on reaction with the trisaccharide
(8) 1131.
Insofar as the complex formed by tri-NAG and the
enzyme is stable, it differs from the enzyme-substrate
complex involved in catalysis. Furthermore, it is known
that at low concentrations, tri-NAG is rather an
inhibitor than a substrate that is broken down. As triNAG fills only half of the cleft, the presence of more
sugar residues, filling the remainder, would appear
[19] L. N. Johnson and D . C. Phillips, Nature (London) 206, 761
(1965).
I201 C. C. F. Blake, L. N. Johnson, G . A. Muir, A. C. T . North,
D . C. Phillips, and V . R . Sarma, Proc. Roy. SOC.(London), Ser. B
167, 378 (1967).
229
necessary for the formation of a reactive enzymesubstrate complex. In fact, another three sugar residues can be added to the tri-NAG in such a way that
satisfactory interactions can take place between the
atoms of the substrate and those of the enzyme[131.
However, the forth sugar residue must be distorted a
little out of its most stable “chair” conformation into
a conformation in which its carbon atoms 1, 2, and 5
and the oxygen atom attached to C-5 all lie in a
plane“31. The distortion assists in breaking the substrate below the fourth sugar. This observation and
many other results indicate that the linkage which is
broken is the one between the fourth and the fifth sugar
residue (between C-1 and the oxygen in the glycosidic
linkage). The most reactive groups in the vicinity of
this bond are the side chains of Asp 52 and Glu 35.
residue becomes distorted and takes up a conformation favoring formation of a carbonium ion.
According to observations by Phillips “31, Blake et
al. [2OJ, and Vernon [211, the events leading to the rupture of the bacterial cell wall probably take the following course:
“For the first time we have been able to interpret the catalytic
activity of an enzyme in stereochemical terms. It seems rash
to generalize from the structure of one enzyme, but we may
broaden our base by elevating myoglobin and haemoglobin
to the rank of honorary enzymes, as Monod has suggested,
and call the heme groups their active sites. What these three
proteins have in common is the distribution of polar and
non-polar side chains; the interior of the lysozyme as well as
that of the globin chains is made up largely of hydrocarbons,
providing a medium of low dielectric constant. In each case
the active center lies in a pocket formed by this non-polar
medium, and the pocket contains some polar residues which
are essential for activity.”
1)The substrate becomes attached to the enzyme and is
held in position by hydrogen bonds and by other
forces; in the process the ring of the fourth sugar
2) A proton is transferred to the glycosidic oxygen
atom from the side chain of Glu 35.
3) Heterolysis of the C1 carbon-oxygen bond gives a
carbonium ion which is stabilized by interaction with
the negative charge of the side chain of Asp 52.
4) The disaccharide obtained from the hexasaccharide
diffuses away and a water molecule attacks the carbonium ion, thus completing hydrolysis (Fig. 4).
St is apparent that three factors are responsible for
the catalytic effect: general acid-catalysis; activation
of the substrate by distortion; and electrostatic interaction.
At this stage, it is fitting to quote a remark made by Perufz [22J:
2.4. Biosynthesis of Lysozyme
The incubation of minced hen oviduct with 3H-leucine
was undertaken by Canfield and Anfinsen 1231, and led
to the formation of radioactive lysozyme. The distribution of radioactivity in the leucine residues along the
polypeptide chain of the reduced, carboxymethylated
protein was established by isolation of leucine-containing peptides produced by trypsin digestion. The
peptides were then hydrolyzed and the specific radioactivities of the leucine residues were determined.
Samples of radioactive lysozyme isolated from oviduct
minces that had been incubated for 3 and 5 minutes,
showed unequal labeling of leucine residues, and the
relative specific radioactivities were found to increase
from the NH2-terminal to the COOH-terminal end of
the chain.
Lysozyme main chain
1168611
Lysozyrne
main chain
Fig. 4. Splitting of substrate by lysozyme is believed to involve the
proximity and activity of two side chains, Glu 35 and Asp 52. A proton
probably becomes detached from the OH group of residue 35 and attaches itself t o the oxygen atom that joins rings D and E, thus breaking
the bond between the two rings 113).
f2lJ C. A. Vernon, Proc. Roy. SOC.(London), Ser. B 167, 389
(1967).
230
These observations [231 are consistent with the model
of amino acid assembly in which the polypeptide
chain grows unidirectionally, initiation occurring at
the NHz-terminal end and termination at the COOHterminal end. St seems reasonable to assurne[l31 that
the folding of the protein chain to its native conformation begins at the amino end even before synthesis is
complete. Parts of the polypeptide chain, particularly
those near the terminal amino end, may fold into
stable conformations that can still be recognized in the
finished molecule and that act as “internal templates”,
or centers around which the rest of the chain is
[22] M. F. Perurz, Proc. Roy. SOC. (London), Ser. B 167, 448
(1967).
[23] R. E. Canfield and C. E. Anfinsen, Biochemistry 2, 1073
(1963).
Angew. Chem. internat. Edit. 1 Vol. 8 (1969) I NO. 4
According to recent investigations by Kronman et
al. 1371 N-bromosuccinimide shows no specificity in its
reaction with the tryptophan groups of lysozyme.
Caution must therefore be exercised in the use of this
reagent for determining the extent of “exposure” of
side chains of native proteins.
folded [131. The three-dimensional model provides
support for these ideas, especially as the first residues
at the terminal amino end form a compact structure.
2.5. Relationship between Structure and Activity
2.5.1. T h e NH2- a n d COOH-Terminal S e q u e n c e s
2.5.3. R o l e of t h e H i s t i d i n e R e s i d u e
The N H 2 - and COOH-terminal sequences are split
offby aminopeptidase and carboxypeptidase respectively. The shortened enzymes, des-lysylvaline-lysozyme 1241 and des-arginylleucine-lysozyme 1251 are always active. At low ionic strength the des-arginylleucine-lysozyme is even slightly more active than the
native molecule, whereas at high ionic strength it
proves to be significantly less active.
Numerous investigations on lysozyrnes whose molecules contain only one histidine residue have shown
that this amino acid is not involved in the active site.
Fully active lysozymes containing no histidine have,
in fact, been isolated from duck egg-white 1381.
2.5.4. R o l e of t h e C y s t i n e R e s i d u e s
Hen egg-white lysozyme contains four disulfide
bridges. With the discovery of lysozymes containing
three L391, two 1401, or no 1411 cystine bonds per molecule, it appeared that these latter are not essential for
biological activity. However, Joll2s et al. 1421 showed
that the presence of disulfide bridges is important for
the heat stability of the enzymes. Lysozymes with four
cystine bonds are relatively stable in acidic or neutral
2.5.2. R o l e o f t h e T r y p t o p h a n R e s i d u e s
Hen lysozyme contains six tryptophan residues per
molecule. Recent X-ray studies at a 2
resolution
demonstrated the importance of some of these residues
for the biological activity [8,191. Other studies in this
field were made by chemical and physical methods 119,26,271 (see Table 2).
Table 2. Relationship between the chemical modification of tryptophan residues and the
biological activity of hen egg-white lysozyme.
Number of modified
tryptophan residues
Reagent
N-bromosuccinimide
N-bromosuccinimide
iodine
hydrogen peroxide
2-hydroxy-5-nitrohenzyl bromide
N-bromosuccinimide
5 mole iodine
1 mole iodine
iodine
ozone
Conversion
product
Amino
acid
(no.)
oxindole
probably
oxindole
[a1
[a1
la1
[a1
[a1
[a1
62
108
108,62
108
1
2
N’-formylkynurenine
108
111
[a] Not examined
1241 J . JollPs and P . JollPs, Biochem. biophysic. Res. Commun.
22, 22 (1966).
1251 W. T. Morgan and J. P . Riehm, Biochem. biophysic. Res.
Commun. 30, 50 (1968).
[26] K. Hayashi, T. Zmoto, and M. Funatsu, J. Biochemistry
pokyo] 55, 516 (1964).
I271 S. S. Lehrer and G . D . Fasman, Biochem. biophysic. Res.
Commun. 23, 133 (1966).
I281 I. Bernier and P . JoNPs, C . R. hebd. SCances Acad. Sci. 253,
745 (1961).
[29J G. J. S. Rao and L . K . Ramachandran, Biochim. biophysica
Acta 59, 507 (1962).
[30] F. J . Hartdegen and J. A . Rupley, Biochim. biophysica Acta
92, 625 (1964).
[31] Y. Hachimori, H . Horinishi, K. Kurihara, and K . Shibata,
Biochim. biophysica Acta 93, 346 (1964).
[32] T. Bewley and C . H . Li, Nature (London) 206, 624 (1965).
[33] K. Hayachi, T. Zmoto, G. Funatsu, and M. Funatsu, J. Biochemistry (Tokyo) 58, 227 (1965).
[34] C. C . F. Blake, Proc. Roy. SOC.(London), Ser. B. 167, 435
(1967).
Angew. Chem. internat. Edit.
1 Vot. 8 (19691 1 No. 4
solution when heated for 0.5 or 1 min at 100°C.
Human lysozymes (three cystine residues) are a little
more heat sensitive; when the cystine content drops
to only two residues, as in goose lysozyme, the heat
stability is almost lost.
[35] J . A. Rupley, Proc. Roy. SOC.(London), Ser. B 167, 416
(1967).
[36] A. Previero, M.-A. Coletti-Previero, and P. Joll2s, J. molecular Biol. 24, 261 (1967).
[37] M. J , Kronman, F. M . Robbins, and R . E. Andreotti, Biochim. biophysica Acta 143,462 (1967).
[38] J . JoNPs, G. Spotorno, and P. JolfPs, Nature (London) 208,
1204 (1965).
[39] P. JollPs, D . Charlemagne, J.-F. Petit, A.-C. Maire, and
J. JoZlPs, Bull. SOC.chirn. biol. 47, 2241 (1965).
[401 A.-C. Dianoux and P . JolIt%, Biochim. biophysica Acta 133,
472 (1967).
[41] A. Tsugita and M. Znouye, J. molecular Biol. 37,201 (1968).
1421 J. Joll2s, A.-C. Dianoux, J . Hermann, 3.Niemann, and P .
JoNPs, Biochim. biophysica Acta 128, 568 (1966).
23 1
It has also been established that the lysozyme with the smallest cystine content has the highest specific activity1421. If the
specific activity of hen egg-white lysozyme is set at unity,
then a value of the same order is found for the duck enzymes,
and 3 & 0.5 for the human lysozymes and 5 -C 0.5 for the
goose enzyme. Interaction between the substrate and the
active center of the goose enzyme probably occurs more
easily because this enzyme exhibits a lower degree of folding.
2.5.5. R e a c t i v a t i o n of R e d u c e d L y s o z y m e
Lysozyme fully reduced with P-mercaptoethanol in
S M urea can be reactivated by enzymic and nonenzymic procedures. Enzymic reactivation 143, 441 occurs, without any induction period, by disulfide interchange 1451. Non-enzymic reactivation proceeds by
oxidation of the reduced protein in buffered solutions
containing small amounts of P-mercaptoethanol. The
reoxidized lysozyme has the same tertiary structure as
the native enzyme 146,471. Active lysozyme can also be
regenerated from the mixed disulfide of hen lysozyme
and cystine 1481.
2.5.6. C r o s s - l i n k i n g of L y s o z y m e
The cross-linking of lysozyme has been effected by
reaction with phenol-2,4-disulfonyI chloride. The
cross-linked protein retains its enzymic activity, has
approximately the same molecular weight as native
lysozyme, and, according to optical rotatory dispersion
analysis 1491, has essentially the same conformation.
2.5.7. I r r a d i a t i o n of L y s o z y m e
Crystallized lysozyme has been irradiated with doses
of X-rays between 1 and 100 millirads and with y-rays
from 6OCo[511. Doses of a few millirads have no
measurable effect on activity or on the amino acid
composition of lysozyme. However, increased doses
convert the original protein into a large number of
modified proteins showing an increased or reduced
lysozyme activity. Only X-ray doses of the order of
magnitude of 100mrads produce changes in the
amino acids which can be determined with an Autoanalyzer. These changes involve mainly the sulfurcontaining, the aromatic, and the long chain aliphatic
amino acids [501.
1431 R. F. Goldberger, C . J . Epstein, and C . B. Anfinsen. J. biol.
Chemistry 239, 628, 1406 (1964).
[44] P. Venetianer and F. B. Straub, Biochim. biophysica Acta
67, 166 (1963).
[45] D . Givol, R . F. Goldberger, and C. B. Anfinsen, J. biol.
Chemistry 239, PC 3115 (1964).
1461 C.J. Epstein and R . F. Goldberger, J. biol. Chemistry 238,
1380 (1963).
1471 R. F. Goldberger and C. J. Epstein, J. biol. Chemistry 238,
2988 (1963).
[48] L. Kanarek, R. A . Bradshaw, and R . L. Hill,J. biol. Chemistry 240, PC 2756 (1965).
1491 G. L. Moore and R . A . Day, Science (Washington) 159, 210
(1968).
[50] K . Dose, S. Risi, and H. Rauchfuss, Biophysik 3, 202 (1966).
[5l] A. M . Desai and K . S . Korgaonkar, Radiat. Res. 21, 61
(1964).
232
2.5.8. C o n c l u s i o n
The importance of residues 35 (Glu) and 52 (Asp) and
of some Trp residues for the biological activity led to
a number of synthetic assays. Thus the synthesis of
polypeptides having carboxyl functions in hydrophobic as well as in hydrophilic regions (Phe/Glu =
1/0.3; mean molecular weight 63 650) has been reported 1521; it was claimed that they are able to degrade
the cell walls of M . lysodeikticus. The syntheses of
short sequences of hen lysozyme were reported by
Morley 1531 and Jolt& 1541.
2.6. Application of Physico-Chemical Methods to the
Study of Lysozyme
On interaction with either a substrate or an inhibitor lysozyme displays a red shift in its ultraviolet spectrum and it was
implied from model studies that tryptophan was involved in
this shift 155.561.
In the presence of N-acetylglucosamine, the circular dichroism of lysozyme increases in the aromatic range, a
phenomenon that is also attributed to participation of
tryptophan 1571.
Significant changes in the tryptophan fluorescence can occur
o n binding of appropriate inhibitors 6 8 9 591. Large changes in
the tryptophan fluorescence of lysozyme and of the lysozymetri-N-acetyl-D-glucosamine complex are observed with
changing p H and are associated with changes in the ionization of certain groups of the enzyme[58,60*611.These fluorescence changes occur over a pH range in which the gross
conformation is thought to be invariant. The selective quenching of tryptophan fluorescence in lysozyme by iodide ions is
also observed 1621. Fluorescence changes in ultraviolet-irradiated lysozyme were studied by Churchich [631.
The optical rotatory dispersion of aqueous solutions of
lysozyme has been measured over a wavelength range of
195-600 nm [641. By this technique, questions regarding the
conformation of the enzyme under different conditions
have been investigated [64-671.
[52] V. K . Naithani and M . M. Dhar, Biochem. biophysic. Res.
Commun. 29,368 (1967).
[53] J . S . Morley, unpublished.
(541 P.JoN2s and J. JoII?s, Helv. chim. Acta 51, 980 (1968).
[ 5 5 ] K . T . Hayashi, G . Imoto, and M . Funafsu, J. Biochemistry
(Tokyo) 54, 381 (1963); 55, 516 (1964).
1561 F. W. Dahlquist, L . Jao, and M . Raftery, Proc. nat. Acad.
Sci. USA 56, 26 (1966).
1571 A. N. Glazer and N . S . Simmons, J. Amer. chem. SOC.88,
2335 (1966).
[58] S. S. Lehrer and G. D . Fasman, Biochem. biophysica Res.
Commun. 23,133 (1966).
[59] M . Shinifzky, V. Grisaro, D . M . Chipman, and N. Sharon,
Arch. Biochem. Biophysics 115, 232 (1966).
[60] R. F. Steiner and H. Edelhoch, Nature (London) 192, 873
(1961).
1611 S. S . Lehrer and G . D . Fasman, J. biol. Chemistry 242, 4644
(1967).
[62] S . S. Lehrer, Biochem. biophysic. Res. Commun. 29, 767
(1967).
1631 J . E . Churchich, Biochim. biophysica Acta 120, 406 (1966);
126, 606 (1966).
[64] Y. Tomirnatsu and W. Gaffield, Biopolymers 3, 509 (1965).
[65] C. Tanford and N. S. Otchin, J . molecular Biol. 15, 489
(1966).
[66] K . Ogasahara and K. Hamaguchi, J. Biochemistry (Tokyo)
XII, 61, 199 (1967).
(671 K . Ikeda, K . Hamaguchi, M . Imanichi, and T. Amano, J. Biochemistry (Tokyo) 62, 315 (1967).
Angew. Chem. internat. Edit./ Vol. 8 (1969) /No. 4
Recent progress in the application of NMR t o problems of
protein structure and function has made it possible t o obtain more detailed information on the behavior of enzymes
in solution [6*1. Partial assignments in the low field (aromatic)
region of the lysozyme spectrum, a comparison of several
denatured conformations, and spectral changes resulting
from inhibitor binding were reported 1681.
The elucidation of the tertiary structure of lysozyme has
stimulated various attempts to find stereochemical rules
relating the primary t o the tertiary structure [ @ I ; this approach might be complemented by a statistical analysis of the
amino acid sequences 1701. A method of recognizing oc-helical
segments in the polypeptide chains with known primary
structure is currently being investigated [711.
Sophianopoulos [721 and Antonini “31 found that the enzyme
polymerizes reversibly with increasing p H at 20 O C ; between
p H 5 and 9, the dimer predominates.
Before concluding this section, mention should be made of
the studies by Hamaguchi[651 concerning the effect of different reagents, of p H , and of temperature on the stability and
conformation of lysozyme.
possible further investigations on the specificity of
the enzyme [781. Once it had been shown that lysozyme
acts on colloidal chitin 1161, there followed a number of
reports on the purification of soluble N-acetylglucosamine oligosaccharides of varying chain
lengths[79,80l and of the action of the enzyme on
these compounds [81J (“chitinase activity” EC 3.2.1.14,
of lysozyme).
One of the low-molecular-weight substances that have been
investigated in detail as a substrate for lysozyme is the tetrasaccharide NAG-NAM-NAG-NAM.
Digestion of this
compound into disaccharide has been followed by Sharon [761
by paper chromatography, colorimetry, and polarimetry.
Together with the disaccharide, numerous oligosaccharides
were characterized.
In addition t o hydrolysis, lysozyme also catalyzes
transglycosylation [76,*21; it is feasible that hydrolysis
of the tetrasaccharide does not proceed by direct
cleavage but by a transfer mechanism (Scheme 1).
Transglycosylation reactions were also reported with
short-chain chitin oligosaccharides 183-861.
2.7. Mechanism of Action of Lysozyme
Lysozyme has been used extensively in degrading bacterial walls (see Section 2.3.) [17,18J. The influence of
various chemical modifications of the walls on lysozyme sensitivity were recently studied by Salton (741
(Table 3) and Hara [751. The isolation of a disaccharide
Table 3. The influence of chemical substitution of cell walls on lysozyme
sensitivity [741.
Modification
[
Effect
~~
0-Acetylation (OH)
Esterification (COOH)
Diethylammonium salt formation (COOH)
Deacetylation at nitrogen
N-Succinylation (-NH2)
Reaction with I-fluoro-2,4-dinitrobenzene
(--NH2)
Reaction with 5-dimethylamino-I-naphthalenesulfonyl chloride (DANS) (-NH2)
~~~
Complete inhibition
Partial inhibition
Partial inhibition
Inhibition
Stimulation
No effect
No effect
(NAG-NAM), of two disaccharide peptides, and the
corresponding tetrasaccharides from lysozyme digests
of Micrococcus lysodeikticus cell walls was described
in detail by Sharon [76,771. These compounds have been
used for the study of the enzymic activity of lysozyme.
The synthesis of derivatives of muramic acid made
2 tetraose (D-D)
D-D-D
D-D
+
2 D-D-D
2 D-D-D
--f
Hexaose (D-D-D)
-+ D-D-D-D
+D
-+ D-D-D-D-D
-> D-D-D-D
+ D-D
+ Disaccharide (D)
+D
Scheme 1. Course of the transglycosylation reaction induced by lysozyme [821.
“D” stands for the cell-wall disaccharide isolated from M . lysodeikricus
(NAG-NAM).
Lysozyme can also act as a transferase; thus Sharon [87J
observed that the enzyme is able to transfer NAGNAM residues to a variety of mono- and disaccharide
acceptors (NAG, D-glucose, D-glucosides, etc.) and to
form and cleave $(1+2) glycosidic bonds. N-acetylglucosamine, and closely related oligosaccharides
strongly inhibit the enzymic activity of Iysozyme 1881,
as also do derivatives of D-glucosamine [891. Fluorescence studies show that these inhibitors interact with
the tryptophan residues in the active site of the enzyme
(Table 4).
~
[78] R . W. Jeanloz, Exposes annu. Biochim. med. 27, 45 (1966).
[79] J. A. Rupley, Biochim. biophysica Acta 83, 245 (1964).
[80] R. F. Powning and H. Irzykiewicz, J. Chromatogr. (Amsterdam) 29, 115 (1967).
1811 R. F. Powning and H. Zrzykiewicz, Biochim. biophysica Acta
[68] J. S. Cohen and 0 . Jardetzky in C . Nicolau: Experimental
Methods in Molecular Biology. Wiley, New York, in press;
Proc. nat. Acad. Sci. USA, in press.
[69] A. M . Liquori, J. Polymer Sci. 12, 207 (1966).
[70] J . W. Prothero, Biophysic. J. 6, 367 (1966).
[71] P. F. Periti, G. Quagliarotti, and A. M . Liquori, 3. molecular
Biol. 24, 313 (1967).
[72] A . J. Sophianopoulos and K . E. Van Holde, J. biol. Chemistry
239, 2516 (1964).
[73] M. Bruzzesi, E. Chiancone, and E. Antonini, Biochemistry
(Washington) 4, 1796 (1965).
1741 M. R. J . Salton, Exposes annu. Biochim. m8d. 27, 34 (1966).
[75] S. Hara and Y. Matsushima, J. Biochemistry (Tokyo) 62,118
(1967).
[76] N . Sharon, Proc. Roy. SOC.(London) Ser. B 167,402 (1967).
I771 D . Mirelman and N . Sharon, Biochem. biophysic. Res. Commun. 24, 237 (1966); J . biol. Chemistry 242, 3414 (1967).
Angew. Chem. internat. Edit. 1 Vol. 8 (1969) / No. 4
124, 218 (1966).
[821 N . Sharon, Third International Lysozyme Symposium,
Milan, 1964.
[83] J. A. Rupley, Science (Washington) 150, 382 (1965).
I841 V. I . Maksimov, E. D. Kavermeva. and N. A. Kravrhenko,
Biochimija 30, 1007 (1965).
[851 F. W. Dahlquist and M. Raftery, Nature (London) 213, 625
(1967).
[861 F. W. Dahlquist, L . Jao, and M. Raftery, Proc. nat. Acad.
Sci. USA 56, 26 (1966).
[871 J. J . Pollock, D . M . Chipman, and N. Sharon, Arch. Biochem. Biophysics 120, 135 (1967); Biochem. biophys. Res. Commum, in press (1969).
1881 M . Shinitzky, V. Grisaro, D. M . Chipman, and N . Sharon,
Arch. Biochem. Biophysics 115, 232 (1966).
[891 A. Neuberger and B. M. Wilson, Biochim. biophysica Acta
147, 473 (1967).
233
3. Lysozymes of Different Origins
Table 4. Changes in the position of the emission maximum (Amax) and
in the height of the emission maximum(Q) of lysozyme on addition of an
inhibitory sugar [SS].
Sugar [a]
I
Tetra-NAG
-1
Di-NAG
0.6
Di-NAG-NAM
I
3
3
NAG-NAM pentapeptide
Gal-NAG
7
NAG
7
NAG-NAM
12
NAG-NAM methyl ester
25
Cellobiose
> 100
-10
-10
-10
-10
-10
0
0
0
0
+40
+25
-12
t 7
+
18
- 7
-12
-12
0
Ial The oligosaccharides are B (1 +4) linked.
Ibl A?.max = shift in the emission maximum.
Icl AQ = change of the height of the emission maximum relative to
that of the enzyme alone (= 100%).
Further synthetic lysozyme substrates, which can be
assayed colorimetrically, have been described by
Lowe 1901 and Osawa (911.
Lysozymes are widely distributed enzymes; they are
found in a great number of organs, tissues, and secretions (spleen, kidney, leucocytes, tears, saliva, milk,
serum) of vertebrates. They also occur in invertebrates, bacteria, phages, and plants. Previous studies
with hen egg-white lysozyme led us to attribute the
following properties to this enzyme 111: (a) basic protein; (b) low molecular weight (15000); (c) stability
at acidic p H at higher temperatures; (d) lability at
alkaline pH; (e) lysis of suspensions of Micrococcus
Zysodeikticus; (f) its action on an appropriate substrate
liberates compounds that can be detected by reagents
for reducing sugars and amino sugars. However, our
recent research[941 in this field has shown that this
definition of a Iysozyme must be at least partly modified. A brief survey of the purification, analysis,
structure, and properties of some lysozymes will be
undertaken in the second part of this review.
2.8. Immunological Studies
Tsugita et al. succeeded in isolating four immunologically active peptides from a peptic digest of lysozyme and in elucidating their chemical structures;
these peptides seemed to bear at least one antigenic
determinant [92,92aJ. A peptide obtained by trypsin
degradation, consisting of three peptide sequences
Trp 62 + Arg 68, Asn 74 + Leu 83, and Leu 84 +
Lys 96 linked by two disulfide bonds Cys 64 + Cys 80
and Cys 76 + Cys 94, was also found to exhibit weak
but definite immunological activity. According to
Bonavida et al. [93J, lysozyme treated with cyanogen
bromide retains a large proportion of the immunological features of the native enzyme. The proximity
of the enzyme active center and a n antigenic determinant of lysozyme has also been suggested 1941. Univalent antibodies of low molecular weight (3.5 S)
could be purified by gel-filtration and DEAE-cellulose
chromatography from lysozyme antisera of rabbits [95J. A selective fractionation of anti-lysozyme
antibodies of different determinant specificities and the
formation of antibodies with specificity directed
against a unique region in native lysozyme have
recently been described 195aI.
[90] G. Lowe, G. Sheppard, M . L . Sinnort, and A . Williams, Biochem. J. 104,893 (1967).
[91] T . Osawa and Y. Nakazawa, Biochim. biophysica Acta 130,
56 (1966).
1921 S. Shinka, M . Imanishi, N . Miyagawa, T. Amano, M .
Inouye, and A . Tsugifa, Biken J. (Osaka) 10, 89 (1967).
I92aI For the significance of the N - and C-terminal regions as an
antigenic site, see H . Fujio, M . Imanishi, K . Nishioka, and T .
Amano, Biken J. (Osaka) 11, 207, 219 (1968).
[93] B. Bonavida, A . Miller, and E. Sercarz, Information Exchange Group No. 5, Immunopathology (Jan. 31, 1967).
1941 R . V . Fellenberg and L. Levine, Immunochemistry 4, 363
(1967).
[95] 0. Kuwahara, S . Shinka, M . Iminishi, N . Miyagawa, T.
Mori, and T . Amano, Biken J. (Osaka) 9, 1 (1966).
[95a] R . Arnon, Europ. J. Biochem. (Berlin) 5, 583 (1968);
R. Arnon and M . Sela, Proc. nat. Acad. Sci. USA 59, in press
(1969).
234
3.1. Purification, Analysis, and Structure
3.1.1. T w o D i f f e r e n t L y s o z y m e s i n t h e S a m e
S e c r e t i o n : D u c k E g g - W h i t e Lysozymes[971
During ion-exchange chromatography of a ducklysozyme rich material on Amberlite CG-50, two
(sometimes three) enzymes were characterized [97J, i.e.
duck lysozymes I, 11, and I11 (in the order of elution
from the column). Two of them (I1 and 111) were always found, even when the enzymes were purified
from a single egg of a well defined species; they are
devoid of histidine [97-1001 (Fig. 5).
Duck 11:
H-Lys-Val-Tyr-Ser-Arg-Cys-Glu-Leu-Ala-Ala-Ala-Met-LysH-Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ala-Ala-Ala-Met-LysDuck 111:
H-Lys-Val-Tyr-Gln-Arg-Cys-Glu-Leu-Ala-AIa-Ala-Met-LysHuman milk :H-Lys-Val-Phe-Glu-Arg-Cys-Glu-Leu-Ala-ArgHen:
Arg-Leu-Gly-Leu-Asp-Asn-Tyr-Arg-Gly-Tyr-Ser-Leu-Gly-Asn-Trp-ValArg-His-Gly-Leu-Asp-Asn-Tyr-Arg-Gl~-T~r-Ser-Leu-Gly-Asn-T~-ValArg-Leu-Gly-Leu-Asp-Asn-Tyr-Arg-Gly-Tyr-Ser-Leu-Gly-Asn-Trp-Val-
Cys-Ala-Ala-Asn-Tyr-Glu-Ser-Ser
-Phe -Am-Thr-Gln-Ala-Thr-Asn-ArgCys-Ala-Ala-Lys-Phe-Glu-Ser-Asn-Phe-Asn-Thr-Gln-Ala-Thr-Asn-ArgCys-Ala-Ala-Asn-Tyr-Glu-Ser-Gly-Phe
-Am-Thr-Glo-Ala-Thr-Asn- Arg-Trp-Glu-Ser-Gly-Tyr -Am-Thr-ArgAsp-Thr-Asn-Gly-Ser-Thr-Asx-TyrAsp-Thr-Asn-GIy-Ser-Thr-Asp-Tyr-
Asp-Thr-Asn-Gly-Ser-Thr-Asx-Tyr-Ser-Thr-Asp-TyrFig. 5. Chemical structures of the N-terminal moities (53 amino acids)
of duck egg-white lysozymes I1 and 111. Comparison with hen egg-white
lysozyme [loo]. N-terminal sequence and peptides in the active site (Glu
35 and Asp 52) of human milk lysozyme 11071. Asx denotes Asp or
Asn.
[96] P . JollPs, Bull. SOC.Chim. biol. 49, 1001 (1967)
[97] J. JolZPs, G. Spotorno, and P. JollPs, Nature (London) 208,
1204 (1965).
1981 M . Imanishi, S. Shinka, N. Miyagawa, T. Amano, and A .
Tsugifa, Biken J. (Osaka) 9, 107 (1966).
1991 J. JoIIPs, J. Hermann, B. Niemann, and P. J o l k , Europ. I.
Biochem. (Berlin) 1, 344 (1967).
[lo01 B. Niemann, J. Hermann, and J . JollPs, Bull. SOC. Chim.
biol. 50, 923 (1968).
Angew. Chew. internat. Edit. J Vol. 8 (1969) 1 No. 4
Thus it was possible, for the first time, to isolate from
the same secretion two Iysozymes which not only exhibit different chromatographic behavior but also
have slight structural differences 199,1001, which cannot
be deduced from a simple comparison of the amino
acid compositions. However, these slight differences
do give rise to differences in reaction velocities, in the
behavior towards a competitive inhibitor such as
NAG [101J, and in the apparent affinity constants for
the bacterial substrate [lo*] (Table 5).
3.1.2. A n U n u s u a l B i r d L y s o z y m e : G o o s e E g g W h i t e Lysozymer4ol
Only one lysozyme could be isolated from geese of
different species and characterized by ion exchange
chromatography. Its amino acid composition is
quite different from those of other bird egg-white lysozymes [40,1031. The low cystine (two residues/molecule) and tryptophan (three residues/molecule) contents [lo41 are of special importance. Goose lysozyme
is very labile at higher temperatures, even at acidic
p H flo1l; its reaction velocity against M . Zysodeikficus
cells rlO2l and its specificity toward low molecular
weight substrates at p H 6.2 are very different from
those of other lysozymes [104,1051.
3.1.3. L y s o z y m e s f r o m N o r m a l H u m a n
Tissues a n d Secretions
JollPs et al. [39,961 obtained a great number of lysozymes from normal human tissues and secretions in a
chromatographically pure state by ion exchange
chromatography on Amberlite CG-50, i.e. lysozymes
from milk [106,1071, tears [1071, saliva [1081, placenta 11091,
spleen, serum, and leucocytes [1101. All these human
enzymes exhibit a very similar behavior on chromatography while differing considerably from bird lysozymes. In this connection it is worth mentioning that
two hen lysozymes (egg-white and lung) were found
to show widely diverging chromatographic behavior;
[loll P. Joll2s, J . Saint-Blancard, D . Charlemagne, A.-C. Dianoux, J. Joll&, and J.-L. Le Baron, Biochim. biophysica Acta 151,
532 (1968).
[lo21 J.-P. Locquet, J. Saint-Blancard, and P. JoMs, Biochim.
biophysica Acta 167, 150 (1968).
[lo31 R . E. Canfield and S. McMurry, Biochem. biophysic. Res.
Commun. 26, 38 (1967).
[I041 A,-C. Dianoux and P. JollPs, Helv. chim. Acta 52, in press
(1969).
rlOS] D . Charlemagne and P. JolPs, Bull. SOC.Chim. biol. 49,
1103 (1967).
I1061 P. Joll2s and J . Jolles, Nature (London) 192, 1187 (1961).
[lo71 J. Jol/es and P. JollPs, Biochemistry (Washington) 6, 411
(1967); Bull. SOC.Chim. biol. 50, in press (1968).
I1081 J.-F. Petit and P. JoNPs, Nature (London) 200, 168
(1963).
[lo91 J.-F. Petit, M . Panigel, and P . JoIIZs, Bull. SOC.Chim. biol.
45, 21 1 (1963).
[110] D . Charlemagne and P. JollPs, Nouvelle Rev. franc. Hematol. 6, 355 (1966).
Angew. Chem. internat. Edit. / Vol. 8 (1969) 1 No. 4
the same applies to two dog lysozymes (spleen and kidney) [I].
Amino acid analyses of all the human lysozymes yielded comparable results. These data seem to indicate the probable
identity of the enzyme in all human tissues and secretions.
The analytical results are in agreement with recent observations of Osserman and Lawbr[1111. However, the data concerning the duck enzymes I1 and 111, which embody structural
differences despite very similar analyses, suggest that caution
should be exercised in interpreting the results obtained for
human lysozymes. A comparison of the tryptic peptides of
the reduced carboxymethylated enzymes seems necessary.
Detailed chemical structure studies concerning milk lysozyme
are already in progress [107I.
The peptide maps obtained by electrochromatography from milk and saliva lysozymes seem almost
identical [1071. Research designed to clarify possible
dissimilarities between other human lysozymes is currently in progress. The human lysozymes isolated by
Jolles el al. have all approximately three to four times
the activity of hen egg-white lysozyme (when titrated
with suspensions of M. lysodeikticus at equivalent
concentrations).
3.1.4. L y s o z y m e s f r o m L e u k e m i a P a t i e n t s
In the case of some leukemias, large quantities of
lysozyme(s) are found in the blood [1121 and/or in the
urine [1111. JolZPs et a / . [1121 determined the serum lysozyme concentration in a group of patients with a variety of diseases of hemopoietic tissues. A correlation
was observed between the serum lysozyme level and
the granulocyte count; a possible relationship with the
monocyte count was also suggested. The lysozymes of
serum and leucocytes from patients suffering from
chronic myelogenous leukemia were purified and
analyzed [* 101. Their composition seemed identical
with that of the enzymes from normal tissues. However, on ion-exchange chromatography on Amberlite
CG-50 the main active peak was always followed by a
smaller one; a comparison of the peptide maps of
both enzymes will be necessary to ascertain whether
they are identical or not.
Osserrnan and Lawlor Ill11 purified human serum and
urinary Iysozymes in monocytic and monomyelocytic
leukemias and found a close correspondence with the
values found for healthy human beings [1071. JollPs confirmed, by an automated method 11131, that only those
leukemia patients suffering from an acute myeloblastic leukemia excrete large quantities of lysozyme;
during purification of the enzyme two biologically
active peaks were again observed on ion-exchange
chromatography [1141. Their specific activities were
higher than those from normal human lysozymes.
[I 111 E. F. Osserman and D. P. Lawlor, J . exp. Medicine I24, 921
(1966).
[I 121 P. JollPs, M . Sternberg, and C. Math&,Third International
Lysozyme Symposium, Milan 1964.
[I131 P . Jollh, M . Bonnafi, A . Mouton, and L . Schwarzenberg,
Rev. franq. etudes clin. biol. 12, 996 (1967).
[114] A . Mouton and P. Joll&, unpublished (1968).
235
3.1.6. P h a g e L y s o z y m e s : A C o n t r i b u t i o n t o
t h e S t u d y of t h e G e n e t i c C o d e . F i r s t in V i t r o
S y n t h e s i s of a n E n z y m e
Escherichia coil cells produce a lysozyme after infection with bacteriophage [1151. Since this process is
controlled by phage DNA, various mutants of lysozyme can easily be obtained by treating phage with
mutagens [1161. Some important problems regarding
this lysozyme in the in vitro system has the characteristics of a de novo synthesis, which is mRNA specific
and is inhibited by chloramphenicol, puromycin, and
RNase. The incorporation of amino acids and the
appearance of lysozyme activity have similar kinetics
and a similar dependence on Mg. RNA from a T4
mutant with an amber mutation in the lysozyme
structural gene is unable to stimulate the synthesis of
lysozyme, thus showing that the measured activity is
that of phage lysozyme [120,1211.
H-Met-Asn-Ile-Phe-Glu-Met-Leu-Arg-Ile-Asp-Glu-Gly-Leu-Arg-LeuLys-Ile-Tyr-Lys-Asp-Thr-Glu-G~y-Tyr-Tyr-Thr-Ile-Gly-I~e-G~y-His-
3.1.7. Lysozyrnes f r o m I n v e r t e b r a t e s
Leu-Leu-Thr-Lys-Ser-Pro-Ser-Leu-Asn-Ala-Ala-Lys-Ser-Glu-Leu-AspLys-Ala-Ile-Gly-Arg-Asn-Cys-Asn-Gly-Val-Ile-Thr-Lys-Asp-Glu-AlaGlu-Lys-Leu-Phe-Asn-Gln-Asp-Val-Asp-Ala-Ala-Val-Arg-Gly-Ile-Leu-Joll2s et al. U23-1241 have found traces of lysozyme(s)
Arg-Asn-Ala-Lys-Leu-Lys-Pro-Val-Tyr-Asp-Ser-Leu-Asp-Ala-~al-Argin many species of invertebrates; annelids, molluscs,
Arg-Cys-Ala-Leu-Ile-Asn-Met-Val-Phe-GInMet-Gly- Glu-Thr-Gly-Valand echinoderms contain larger quantities. Of special
Ala-Gly-Phe-Thr-Asn-Ser-Leu-Arg-Met-Leu-Fln-Gln-Lys-Arg-Trpinterest are the observed seasonal variations of the
Asp-GIu-Ala-Ala-Val-Asn-Leu-Ala-Lys-Ser-Arg-Trp-Tyr-Asn-Gln-Thramount
of enzyme[1251, as well as the fact that these
Pro-Asn-Arg-Ala-Lrs-Arg-Val-Ile-Thr-Thr-Phe-Ar~-Thr-Gly-Thr-TrpAsp-Ala-Tyr-Lys-Asn-Leu-OH
Fig. 6. The amino acids equence of T4 phage lysozyme 1411. The sequence of T 2 phage lysozyme is the same with 3 substitutions: Asn (40)
+ Ser, Ala (41) + Val, T h r (151) + Ala [115a].
the genetic code may well prove solvable by comparing
the chemical structure of mutated lysozymes with that
of wild type lysozyme. By use of acridines such as
proflavine, which is known as a frame-shift mutagen11171, one can determine not only the absolute
RNA code words for amino acids in vivo but also the
direction of reading of lysozyme messenger RNA and
its nucleotide sequence. Tsiigita ef al. [1181 have already determined 76 absolute code words for eleven
amino acids, demonstrated the direction of messenger
RNA reading, and elucidated a part of the nucleotide
sequence of the messenger R N A of T4 phage lysozyme from a comparative study of the chemical
structure of the proteins of wild type and double proflavine-induced mutants.
The complete amino acid sequence of T4 phage lysozyme was also elucidated @ll. The molecular weight
was reported to be ca. 18000. No common primary
structure was found between T4 phage lysozyme and
egg-white lysozyme, but the distribution of basic,
acidic, and hydrophobic amino acids seems to be similar. T4 phage lysozyme contains n o S-S bridge and
only two tryptophan residues per molecule. It is less
stable than the hen enzyme [1191.
Active lysozyme of bacteriophage T4 has been synthesized in a cell free system programmed by RNA
from cells infected with the phage. The appearance of
[115] G. Koch and W. J. Dreyer, Virology 6, 291 (1958).
[115al M . Inouye and A . Tsugita, J. molecular Biol. 37,213 (1968).
[116] G . Streisinger, F. Mukai, W. J . Dreyer, B. Miller, and S .
Horiuchi, Cold Spring Harbor Sympos. quantitat. Biol. 26, 25
(1961).
[117] S. Brenner, L. Barnett, F. H. C. Crick, and A . Orgef, J.
molecular Biol. 3, 121 (1961).
[118] E. Terraghi, Y . Okada, G . Streisinger, J . Emrich, M . Inouye,
and A . Tsugita, Proc. nat. Acad. Sci. USA 56, 500 (1966).
11191 A . Tsugita, M . Inouye, E. Terzaghi, and G . Streisinger,
J. biol. Chemistry 243, 391 (1968).
236
enzymes possess a high chitinase activity. The presence of lysozyme-like substances in insects has been
reported by Mohrig [12*aJ.
3.1.8. L y s o z y m e s f r o m P l a n t s
The presence of bacteriolytic activity in plant tissues
was first reported by Fleming [I41 in 1922. Later Meyer
et al. 11261 reported lysozyme activity in crude proteolytic enzyme preparations from papaya and fig latex.
In 1955, Smith et af. 11271 prepared a crystalline lysozyme from papaya latex and quite recently Howard
and Glazerr1281 studied such a lysozyme in detail. It
was found that papaya lysozyme has a molecular
weight of about 25000, that glycine is its sole aminoterminal residue, and that the molecule consists of a
single polypeptide chain. Papaya lysozyme lyses M .
lysodeikticus cell walls at a rate one-third of that exhibited by egg-white lysozyme; however, it displays a
chitinase activity toward tetra-N-acetyl-D-glucosamine
400 times that of hen lysozyme. No involvement of
tryptophan residues in the active site of papaya lysozyme could be demonstrated. Similar observations
have been made by Joll2s et al.[1221 with a turnip
lysozyme.
[120] R . F. Gesteland, W. Salser, and A. Bolle, Proc. nat. Acad.
Sci. USA 58, 2036 (1967).
[121] R . F. Gesteland, W. Salser, and A. Bolle, Nature (London)
215, 588 (1967).
11221 P. Jolles, M . Horisberger, and E. van Leemputten, unpublished data (1968).
[I231 P. Joll2s, J . Jolf2s-Thaureaux, and C. Fromageot, C. R.
SCances SOC.Biol. Filiales 151, 1368 (1957).
I1241 P. Jollds and S . Zuili, Biochim. biophysica Acta 39, 212
(1960).
[125] B. Niemann and P. Jolles, unpublished (1968).
[125a] W. Mohrig and B. Messner, Biol. 2. 87, 439 (1968).
[126] K. Meyer, E. Hahnel, and A . Steinberg, J. biol. Chemistry
163, 733 (1946).
[127] E. L. Smith, J . R . Kimmel, D . M . Brown, and E. 0. P.
Thompson, J. biol. Chemistry 215, 67 (1955).
[128] J . B. Howardand A. N . GIgzer, J. biol. Chemistry 242, 5715
(1967).
Angew. Chem. internat. Edit.
/ Vol. 8 (1969) / No. 4
3.2. Reaction Velocity, Specificity, and
Mode of Action
Lysozymes from different animal species and sometimes also from different organs or secretions of the
same animal are chemically different but have qualitatively the same biological activity, i.e. they promote
lysis of a suspension of dead cells of Micrococcus
lysodeikiicus (basic definition of a lysozyme) [1291.
Quantitatively the reaction velocity differs from one
enzyme to another; differences in the specificity 196,1051,
behavior toward inhibitorsrlol, 1021, etc., were later
observed. In all these experiments, various substrates
were employed, e.g. dead cells of M. lysodeikticus, cell
walls from lysozyme sensitive bacteria ( B .megatherium),
cell walls from Mycobacteria r1301, soluble substrates
from M. lysodeikticus, polymers of N-acetylglucosamine obtained from chitin (chitotetraose, etc.), and
cell-wall saccharides such as NAG-NAM-NAGNAM.
3.2.1. R e a c t i o n Velocity. A p p a r e n t A f f i n i t y
Constants
Under the same conditions, bird egg-white lysozymes
digest many of the above mentioned substrates more
rapidly than do human lysozymes 196,1291. At pH 6.2,
duck lysozymes seem to have the most complete and
most rapid action on chitotetraose and chitopentaose [105J. Affinity constants of seven different lysozymes for the bacterial substrate are shown in Table
5 [1021.
not to free amino sugars. About 3 0 % of the tetrasaccharide was not hydrolyzed. However, transglycosylation products were observed thus suggesting that
the mode of action of these four lysozymes is the
same [1311. The four lysozymes mentioned digest
tetra-NAG and penta-NAG in a similar manner: the
same digestion products are observed including free
NAG and transglycosylation products. The quantity
of tetra-NAG not digested varies at a given p H from
one enzyme to another [1051.
However, the goose lysozyme behaves differently: at
pH 6.2 M. lysodeikticus cells are digested rapidly but
the lysis soon stops. Low molecular weight substrates,
such as NAG-NAM-NAG-NAM
or chitotetraose
are gradually attacked only at lower pH11051. The
reaction products obtained from the cells also vary
with the pH 11041.
3.2.3. T h e B e h a v i o r of L y s o z y m e s in t h e
P r e s e n c e of a n I n h i b i t o r
It has been shown that hen egg-white lysozyme can be
inhibited by NAG [19,301. Under the same experimental conditions, this inhibitory action varies greatly
from one enzyme to another[lUll. The human lysozymes are more strongly inhibited by NAG than
is hen lysozyme. At p H 6.2, the two duck enzymes are
inhibited to less than 50% and goose lysozyme is
practically uninhibited (Fig. 7). Similar results were
Table 5 . Apparent affinity constants of Iysozymes f r o m different origins for Micrococcus
lysodeikricus cells [102].
Apparent
affinity
constant
(mg cells/l)
Lysozyme
I
Human
normal leucocytes
normal plasma
milk
90 I 10
loo*
1105
5
10
I
I
I
I
10
20
30
LO
[NAG1 (mM)
+
Fig. 7. Inhibition of the action of different lysozymes (0.63
on M . lysodeikticus cells by NAG (at pH 6.2) [IOl]. x
0 = hen, A = duck 11, 0 = duck 111, = goose.
~
Avian
hen egg-white
duck egg-white I1
115*
10
150 f 10
3.2.2. S p e c i f i c i t y a n d M o d e of A c t i o n
The action of hen, duck, and human lysozymes on
the substrates mentioned in Section 3.2 leads not to
the formation of free sugars or amino acids but, in
many cases, to the disaccharide NAG-NAM [96,129J.
Like the hen enzyme, duck lysozymes and human-tear
lysozyme degrade the tetrasaccharide NAG-NAM NAG-NAM to the disaccharide NAG-NAM but
-
~
~
11291 P. Joll&, Proc. Roy. SOC.(London) B 167, 3 5 0 (1967).
11301 H . de Wiis and P. JoI/Cs, Biochim. biophysica Acta 83,
326 (1964).
Angew. Chem. infernat.Edit. Vol. 8 (1969) 1 No. 4
x
10-6~)
human,
obtained with tetra-NAG [loll. Other inhibitors of
hen lysozyme, such as histamine or L-histidine methyl
ester [**I, also inhibit goose lysozyme at concentrations similar to those used for the hen enzyme [ l o l l .
3.2.4. I m m u n o l o g i c a l C o m p a r i s o n of
Lysozymes
Arnheim and Wilson11321 purified lysozyme by Sephadex G-75 chromatography from the egg-whites of
17 species of birds in the order Galliformes and tested
11311 N. Sharon, J . Joll2s, and P. Jollt!~,Bull. SOC.Chim. biol.
48, 731 (1966).
11321 N . Arnheim and A. C . Wilson, J. biol. Chemistry (1968), in
press.
237
for reactivity with antichicken lysozyme. The chicken
and its closest relative, the jungle fowl, have lysozymes
that appear to be indistinguishable from each other.
The lysozymes of all the other species tested (partridges, quails, pheasants) could be distinguished from
the chicken enzyme by quantitative micro-complement
fixation. Immunodiffusion failed to detect most of
these differences in antigenic structure. A particularly
close relationship was observed between the lysozymes
of the chicken, partridge, and American quails.
Osserrnann and Lawlor [ l l l l reported some immunochemical data which suggest the identity of the human
lysozymes studied until now. Dissimilarities to bird
egg-white enzymes were substantiated by the failure
of the antiserum to a human lysozyme to inhibit the
activity of the egg enzyme.
4. Concluding Remarks
4.1. Definition and Significance of Lysozymes
The results described in Section 3 necessitate a slight
revision[961 of the definition of a lysozyme given in
Sections 1 and 2. It is possible to imagine a lysozyme
In vivo lysozyme can be included in an artificial structure
formed by phospholipids and protein that is. by components
common to many biological membranes. capable of masking
its catalytic activity“341. The demonstration of such a
phenomenon may offer a new approach in the study of
enzyme latency and may be of some importance in the
development of a theory of membrane formation.
4.2. The Place of Lysozyme Among the Enzymes
Degrading Peptidoglycans
The enzymes degrading peptidoglycans of bacterial cell walls
are mainly bacteriolytic endoenzymes. They can be grouped
into three classes: carbohydrases, acetylmuramyl-L-alanine
amidases, and peptidases 11351. Carbohydrases include endoacetylmuramidases, endoacetylglucosaminidases, and exoacetylglucosaminidases. Lysozymes belong to the group of
endoacetylmuramidases, which also include the “ 3 2 enzyme”
from Streptomyces albus G “361. the “F1 enzyme” from St.
afbusG 11371, the “B enzyme” from Chalaropsis 11381, and Autolysin from Streptococcus faecalis “393.
It is worth mentioning that lysozyme and penicillin, which
were both discovered by Fleming. react with the samepeptidoglycan from bacterial cell wall. Lysozyme splits the NAMNAG linkage and penicillin majnly prevents biosynthesis of
the peptide bridges. Furthermore, penicillin inhibits the lytic
action of lysozyme, probably because it bears a structural
resemblance to a portion of the bacterial cell wall, N-acetyimuramic acid”@] (Fig. 8).
I
L-LYS
b
with an isoelectric point lower than 10.5-11 (hen
enzyme); the molecular weight can be higher than
15000, as for the phage enzyme (18000)[119J; the
stability at higher temperatures is lost when the cystine
content of the enzyme becomes too low 1421. A property common to all the enzymes studied so far is the
ability to rapidly lyse bacteria such as M. lysodeikticus
by splitting P (1 +4) linkages between a NAM and a
NAG residue. Behavior towards low molecular
weight substrates seems to depend on the pH, at least
for some of the enzymes [1041.
Only few details are avaiIable concerning the role of lysozymes in vivo [1331. Hen egg-white lysozyme is the only lysozyme
yet to have been used clinically, owing to its basic character
(addition to cow milk) and its antibacterial action. The latter
can be explained in terms of enzymic action o n substrates in
the cell walls of gram-positive and gram-negative bacteria,
although the substrates are not easily accessible in the gramnegative type. In vivo in the presence of substances such as
enzymes, complement fractions, immunoglobulins [133aj, or
nucleic acids [133b], lysozyme seems to have some additional
properties that have not yet been studied.
[133] P. Jollt?s, Revue des Docteurs en Pharmacie (Paris) 1968.
[133a] M . W . Smith, R . Witty, and P . Brown, Nature (London)
220, 387 (1968).
[133b] D . Cattan, D. Bourgoin, and M. Joly, Bull. SOC.chim.
biol. 50, 409 (1968).
238
Fig. 8 . A fragment of the peptidoglycan of Staphylococcus aureus showing point of cleavage by various enzymes 11351. X = NAG; Y = NAM;
A = lysozyme; B = endoacetylglucosaminidase; C = acetylmuramyl-Lalanine amidase; D and I = endopeptidases.
4.3. Lysozyme and Evolution
Lactose synthetase (EC 2.4.1 .c) catalyzes the following
reaction [1411:
UDP-D-galactose
+ CC-D-glUCOSe
+ lactose
+
UDP
[134] D . Romeo and B. de Bernard, Nature (London) 212, 1491
(1966).
11351 J . L . Strominger and J.-M. Ghuysen, Science (Washington)
156, 213 (1967).
[136] J. M. Ghuysen and J. L. Strominger, Biochemistry (Washington) 2, 1110, 1119 (1963).
[137] E. Munoz, J . M . Ghuysen, M . Leyh-Bouille, J.-F. Petit, and
R. Tinelfi, Biochemistry 5, 3091 (1966).
[138] D . J . Tipper, J L. Strominger, and J. M . Ghuysen, Science
(Washington) 146, 781 (1964).
I1391 M . J . Conover, J. S. Thompson, and G. D . Shockman, Biochem. biophysic. Res. Commun. 23, 713 (1966).
[140] H . Felsenfeld and R. E. Handschumacher, Molecular Pharmacol. 3, 153 (1967).
[141] U.Brodbeck, W . L . Denton, N . Tanahashi, and K . E. Ebner,
J. biol. Chemistry 242, 1391 (1966).
Angew. Chem. internat. Edit. 1 Vol. 8 (1969)/No. 4
The soluble enzyme from milk requires the presence
of two proteins, A and B, for activity; individually
these proteins do not exhibit any catalytic activity.
The present view is that A and B proteins are naturally
occurring subunits and that enzymic activity is dependent upon the formation of an AB complex. The B
protein was crystallized and identified as a-lactalbumin. Its structure was also established (Fig. 9). It
of the mammary gland. a-Lactalbumin also seemed to
be involved in reactions leading to the synthesis of
p(1+4) linkages whereas lysozyme hydrolyzed such
bonds. However, more recently the B protein appeared
to act as a substrate modifier, changing the specificity
of the A protein (inhibition of Gal-NAG synthesis in
favor of Gal-Glc synthesis). Furthermore, a genetic
relationship also exists between avidins and lysozymes 11431.
Although lysozyme and a-lactalbumin have similar
chemical structures [I421 the latter has no lytic activity,
Ala-Met-Lys-Arg-His-Gly-Leu-Asp-Asn-Tyr-Arg-Gly-Tyr-Ser-Leu-Gly-probably because residue 35 (Glu) of hen lysozyme,
GIu-Leu-LysAsp-Leu-Lys- Gly-Tyr-Gly-Gly-Val-Ser-Leu-Pro- which is important for the catalytic activity, is replaced by a hystidine residue.
Asn-Trp-Val-Cys-Ala-Ala-L ys-Phe-Glu-Ser-Asn
-Phe-Asn-Thr-GlnH e n lysozyme:
H-Lys-Val-Phe-Gly-Arg-Cys-Glu-Leu-Ala-AlaBovine a-lactalbumin: H-Glu-Glu-Leu-Thr-Lys-Cys-Glu-Val-Phe-Arg-
Glu-Trp-Val-Cys-Thr-Thr-
Phe-His-Thr-Ser-Gly-Tyr -Asp-Thr-Glx-
However, despite the fact that hen egg-white-, phage-,
and papaya-latex lysozymes have entirely different
chemical structures, they all promote lysis of M. iysoIle-Asn-Ser-Arg-Trp-Trp-Cys-Asn-Asp-Gly-Arg-Thr-Pro-Gly-Ser-Arg-deikticw cells. The main function of hen egg-white
Ile,Asx,Asx)-Lys-Ile-Trp- Cys- Lys- Asx-Asx-Glx-Asx-Pro-His-Ser-Serlysozyme, other bird lysozymes (even the goose enA s n - L e u - Cys- Asn-IIe-Pro-Cys-Ser-Ala-Leu-Leu-Ser-Ser-Asp-Ile-Thrzyme with its quite different behavior), human lysoAsx-Ile-Cys - Asn-He -Ser-Cys-Asp-Lys-Phe-Leu- Asx-Asx- Asx-Leu-Thrzymes (rnol. wt. 15000) [96.1071, etc., which have simiAla-Ser-Val-A s n -Cys-Ala-Lys-Lys-Ile-Val-Ser-Asp-Gly- Asp-Gly-Metlar structures and active sites (Fig. 5 ) , seems to be
(Asx,Asx,IIe)-Met-Cys-Val-Lys-Lys-IIe-Leu- Asp-Lys-Val-Gly-Iledigestion of bacterial cell walls, whereas phage lysoAsn-Ala-Trp-Val-Ala-Trp-Arg-Asn-Arg-Cys-Lys-Gly-Thr-Asp-Val-Glnzyme (mol. wt. 1 8 0 0 0 ) [ 1 1 9 1 seems to exhibit a specific
Asn-Tyr-Trp-Leu-Ala-His-Lys-Ala-Leu-Cys-Ser-Glu-Lys-Leu-Asp-Glnaffinity for E. coii cells. On the other hand, papayaAla-Trp-Ile-Arg-GIy-Cys-Arg-Leu-OH
latex lysozyme (mol. wt. 25 000) [1281 is much more a
-Trp-LeuCys-Glu-Lys-Leu-OH
chitinase than a lysozyme and the tryptophan residues
n o longer seem to be involved in the active site. This
Fig. 9. A m i n o acid sequence of bovine a-lactalbumin compared with
the sequence of hen egg-white lysozyme [1421. Asx stands for A s p or
is also true for turnip lysozyme and lysozymes from
Asn: Glx for Glu or Gln.
invertebrates. The question thus arises whether such
enzymes can still be called lysozymes, which should
primarily
be muramidases (lysozyme = N-acetylmurshows that a-lactalbumin has a close structural simiamylhydrolase)
with slight chitinase activity. The
larity to hen egg-white lysozyme. According to Brew
chitinase activity must be present, since an enzyme
et aZ.11421, the wide distribution of Iysozyme in the
obtained from Chaluropsis which has only muramidase
animal kingdom taken in conjunction with the unique
activity was not called lysozyme Ll441.
occurrence of a-lactalbumin and lactose in milk suggests that the direct evolution of a-lactalbumin from
Received: June 19, 1968
[ A 686 IE]
a lysozyme in a n ancestor of the mammals was a
G e r m a n version: Angew. C h e m . 81, 224 (1969)
prime step in the evolution of lactose synthetase and
Ala-Thr-Asn-Arg-Asn-Thr-Asp-Gly
- Ser-Thr-Asp -Tyr- Gly - Ile-Leu-Gln,
(Ala,Ile,Val,Glx)-Asx-Asx-(Glx,Ser,Thr,Asx)-Tyr-Gly-Leu-Phe-(Glx-
[142] K. Brew, T. C. Vanaman, and R . L . Hi",J. biol. Chemistry
242, 3741 (1967).
Angew. Chem. internat. Edit. j Vol. 8 (1969) 1No. 4
[143] N . M . Green, Nature (London) 217, 254 (1968).
11441 J . H . Hush and M . V. Rorhluuf, J. biol. Chemistry 242,
5586 (1967).
239
Документ
Категория
Без категории
Просмотров
0
Размер файла
1 299 Кб
Теги
chapter, molecular, lysozyme, biologya
1/--страниц
Пожаловаться на содержимое документа