ROLE OF THE PYRROLIDINE RING OF PROLINE IN DETERMINING THE SUBSTRATE-SPECIFICITY OF CDC2 KINASE OR CDK5

To examine structural features of proline which are essential for the proline-directed phosphorylation by cdc2 kinase or cdk5, we prepared t he peptide representing the cdc2 kinase phosphorylation site at Ser-55 in vimentin -Ser-Ser-Ser(55)-Pro(56)-Gly-Gly(58)-Ala-Tyr-NH2], the pe ptide containing arginine in place of Gly-58, and their derivatives co ntaining various N-methylamino acids or proline homologs in place of P ro-BE, and tested them as substrates for the kinases, While substituti on of the proline by proline homologs (L-pipecolic acid or L-azetidine -2-carboxylic acid) increased the K-m value 2- to ii-fold at utmost, s ubstitution by N-methylamino acids (sarcosine, L-N-methylalanine, L-N- methylvaline, or L-N-methylleucine) increased the K-m value 7- to 40-f old for cdc2 kinase, For cdk5, these substitutions led to parallel eff ects on the K-m value to those found for cdc2 kinase; cdk5 recognized the peptides with a proline specificity similar to that for cdc2 kinas e, These results suggest that the pyrrolidine ring of proline is impor tant for substrate recognition by cdc2 kinase or cdk5, Molecular dynam ics and molecular mechanics simulations indicated that the pyrrolidine ring of proline is optimal to stabilize a beta-turn at the phosphoryl ation site and that the K-m values of the peptides for the enzymes mig ht be related to the probability of the turn structure, The results ob tained here also suggest that the pyrrolidine ring of proline is requi red to maintain a high V-max value for cdc2 kinase or especially for c dk5, These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.