The literature on the above subject is very meager but interestingly enough what little
is available is in good empirical agreement with the relative rates of hydrolysis
of the peptide bonds of those peptides which have been isolated from lysosome: in
the case of a regular structure such as that postulated for collagen and gelatin,
one might anticipate the breaking of particular bonds.
I should like to present the information in the literature and in our own data for
your consideration because I believe that if it were possible to decide what are the
causes of the differences in the ease of hydrolysis of various peptide bonds it would
greatly aid our studies of protein structure.
Literature
In studying the hydrolysis of gramicidin, Christensen (J. Biol. Chem., 151, 319 (1943)
and 154, 427 (1944)) observed that the hydrolysis of valylvaline was very difficult
even in refuxing hydrochloric acid. Synge (Biochem. J., 44, 542 (1949)) substantiated
this work but earlier (Biochem. J., 39, 351 (1945)) he had studied the relative rates
of hydrolysis in acid at 37° of a number of peptides. He found that glycyl peptides
(for example, glycylleucine, glycylglycine was more difficult and valylglycine was
very difficult to hydrolyze. Form these results, the R group attached to that amino
acid which contributes the –CO- to the peptide bond appears to play an important role.
Christensen and Hegsted (J. Biol. Chem., 158, 593 (1945)) made a further study of
the hydrolysis of the various peptide bonds in gramicidin at 37° and at refluxing
temperatures and concluded that the release of valine is slow, that the release of
tryptophan is more rapid than that of the others except at 37°, that linkages with
alanine are relatively stable at both temperatures, and that, when leucine contributes
–CO- to the peptide bond, the bond is relatively stable to boiling.
Desmielle and Casal (Biochim. Biophys. Acta, 2, 64 (1948)) have demonstrated that
a very labile bond results when serine or threonine contributes –NH- to the peptide
bond and they have evidence that the following shift takes place under the influence
of strong acid: [Figure of Chemical Reaction Step]
Our Work
In our attempt to produce longer end peptides from DNP-lysozyme a study was made of
the nature of the peptide mixture which is obtained by refluxing in 6 N hydrochloric
acid for various periods. The results are shown in Figure 1. Analysis of the peptides
established the sequence pictured in Figure 2. The conclusion may be drawn from these
data that the relative rate of fission of the bonds is
3 > 4 > 1 > 2 > 5
Partial hydrolysis of each of the peptides substantiates these conclusions.
It has also been possible to recognize seryl and threonyl peptides: in one- experiment,
lysozyme itself was partially hydrolyzed and the partial hydrolysate was dinitrophenylated.
From the mixture, seryl and threonyl peptides were isolated end their composition
may have been seryl (leucine, alanine, glycine) in which the sequence of leucine,
alanine, and glycine is undetermined and threonylaspartic acid.
Remarks
The information in the literature would lead us to expect that Bond 2 would be a relatively
stable one and Bond 4 a relatively labile one and this is found to be the case. No
data about bonds like 1 and 3 seem to be available. Bond 5 may be very stable or Bond
4 may be a very labile one if the fifth amino acid should be serine or threonine or
perhaps tryptophan.
The isolation of seryl and threonyl peptides provides further proof of the work of
Desnuelle and Casal. If the seryl peptide should be seryl (leucine, alenine) glycine,
this fact would indicate again the lability of bonds in which glycine provides –CO-.
In attempting to explain the observed facts, one immediately thinks of the possibilities
of steric hindrance. The use of the models shows that in an extended polypeptide chain
the R group of an amino acid in certain positions could protect one side of the bond
to which its amino acid contributes -CO- but that the other side would be open to
the hydrolyzing medium unless protected by the R group of the amino acid which provides
-NH- to the bond. On this basis, the stability of valylglycine toward hydrolysis is
difficult to explain. Of course, if hydrolysis requires the approach of the hydrolyzing
molecule from a certain direction, the protection furnished by one R group may have
significance.
On the other hand, in the 3,7—residue helix the R group can provide no protection
to that bond to which the amino acid contributes -CO- but it can protect, that to
which it contributes -NH-.
Perhaps the differences in the ease of hydrolysis of the various peptide bonds is
associated with the resonating character of the bond but it is somewhat difficult
to see how the R groups would influence it.
I would greatly appreciate any comments which you would care to make on this problem.
