Role of enzyme-peptide substrate backbone hydrogen bonding in determining protein kinase substrate specificities

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Abstract
As part of a search for peptides that have specificity for selected protein kinases, the possibility that adenosine cyclic 3'',5''-phosphate dependent protein kinase (A-kinase) recognizes the hydrogen-bonding potential of its peptide substrates was investigated. A-kinase catalyzes the phosphorylation of five N.alpha.-methylated and four depsipeptide derivatives of Leu-Arg-Arg-Ala-Ser-Leu-Gly (peptide 1) at rates that differ by at least 7 orders of magnitude. These peptide 1 analogues each lack the ability to donate a hydrogen bond at selected positions in the peptide chain. If a particular amide hydrogen of a peptide amide is involved in hydrogen bonding, which is important for enzyme recognition, the prediction is that peptides which contain an ester or a N-methylated bond at that position in peptide 1 will be comparatively poor substrates. In contrast, if a depsipeptide has a reactivity comparable to that of peptide 1 but the analogous N-methylated peptide has a poor reactivity with A-kinase, the result might indicate that the N-methyl group causes unfavorable steric effects. The depsipeptide that lacks a Leu6 amide proton is a good substrate for A-kinase, but the corresponding N-methylated peptide is phosphorylated far less efficiently. This result and others presented in this paper suggest that although enzyme-substrate hydrogen bonding may play some role in A-kinase catalysis of phosphoryl group transfer, other explanations are necessary to account for the relative reactivities of N.alpha.-methylated and depsi-containing peptide 1 analogues. Alternate explanations that cannot be eliminated from the data presented here include the presence of disruptive peptide-enzyme steric interactions or intrapeptide steric interactions that might prevent a peptide 1 analogue from assuming a conformation recognizable by A-kinase. These possibilities are examined further in the following papers.