Role of the Pyrrolidine Ring of Proline in Determining the Substrate Specificity of cdc2 Kinase or cdk5

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To examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared the peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin [Ser-Leu-Tyr-Ser-Ser-Ser⁵⁵-Pro⁵⁶-Gly-Gly⁵⁸-Ala-Tyr-NH₂], the peptide containing arginine in place of Gly-58, and their derivatives containing various N-methylamino acids or proline homologs in place of Pro-56, and tested them as substrates for the kinases. While substitution of the proline by proline homologs (L-pipecolic acid or L-azetidine-2-carboxylic acid) increased the K_m value 2- to 4-fold at utmost, substitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N-methylvaline, or L-N-methylleucine) increased the K_m value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the K_m value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinase. These results suggest that the pyrrolidine ring of proline is important for substrate recognition by cdc2 kinase or cdk5. Molecular dynamics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a β-turn at the phosphorylation site and that the K_m values of the peptides for the enzymes might be related to the probability of the turn structure. The results obtained here also suggest that the pyrrolidine ring of proline is required to maintain a high V_max value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.