Substrate Specificity of Protein Kinase C Studied with Peptides Containing D-Amino Acid Residues1

Abstract

A set of stereoisomeric nonapeptides KRPSQRAKY with one, two, or all L-amino acid residues replaced by the corresponding D-amino acids, and two analogs with L- and D-threonine instead of serine, were synthesized and tested as substrates for protein kinase C. All of the peptides were phosphorylated by the enzyme. The maximal rate of the reaction with the all-D peptide was more than one order of magnitude lower than that for all-L peptide with serine. The same applied to the peptides with D-Ser or with D-Arg in position +2 with respect to Ser. The Km values for the peptides containing one D-amino acid were close to that for the prototype peptide (53 microM). On the other hand, when two or more D-amino acids were present, the Km value increased considerably. Replacement of serine by threonine also reduced the phosphorylation rate and increased the Km values. One can conclude that the stereospecificity of protein kinase C is much less pronounced than that of protein kinase A, which is in agreement with the less clearly pronounced substrate specificity of the former enzyme.

Inspiration from the mirror: D-amino acid containing peptides in biomedical approaches

“…Then, we discuss the less expected enzyme reactions of D-amino acid, especially highlighting the recent study of D-amino acids for enzyme-instructed self-assembly (EISA), which results in selectivity in potential anti-inflammatory or anticancer drug approaches. We hope that these discussions will help readers to appreciate the study of D-amino acids in achieving biostability(17, 18), investigating interactions between enzymes and D-peptides(19, 20), and improving bioactivities via the introduction of D-amino acids(21, 22). …”

The specificity of the protein kinase C α, βII and γ isoforms as assessed by an unnatural alcohol-appended peptide library

“…PKC catalyses the phosphorylation of -serine as well as configurationally related analogues in conventional and Cterminally derivatized peptides [11,23]. This pattern holds for Nterminus-appended serine residue as well.…”

Quantitative Structure–Activity Relationships in the Protein Kinase C Reaction with Synthetic Peptides Derived from Myelin Basic Protein

“…On the other hand, introduction of the independent variable for charged groups significantly improved the quality of the correlations, including the relationship for log V. Correlation of the latter parameters simultaneously with ȏ, MR, and Ind yielded log V ϭ Ϫ(7.25 Ϯ 0.11) Ϫ (0.02 Ϯ 0.04)ȏ ϩ (0.013 Ϯ 0.006)MR ϩ (0.32 Ϯ 0.08)Ind, n ϭ 13, s ϭ 0.147, r ϭ 0.898. [6] It can be seen that hydrophobicity as well as bulkiness of substituents did not play a significant role if compared with the effect of ionic charge in the latter equation, and thus the relationship can be simplified to a single-parameter correlation equation, log V ϭ Ϫ(7.01 Ϯ 0.05) ϩ (0.41 Ϯ 0.09)Ind, [7] n ϭ 13, s ϭ 0.170, r ϭ 0.825.…”

“…In summary, the structure-activity relationships, presented by the best correlation equations [2], [3], and [6] (or [7]), can be taken as descriptors of specificity of protein kinase C against variable structure of peptide substrates in the position directly following the phosphorylatable serine residue.…”