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Memorandum from Walter Schroeder to Linus Pauling. October 30, 1951.
Schroeder writes to present the results of his literature review on the topic of "relative rate of hydrolysis of peptide bonds as a function of the amino acids joined by such a bond."

Transcript

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.

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