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

• Published in: Journal of biochemistry (Tokyo) Vol. 122; no. 2; pp. 409 - 414
• Main Authors: Ando, Shoji, Ikuhara, Toshihiko, Kamata, Tomoko, Sasaki, Yasuharu, Hisanaga, Shin-ichi, Kishimoto, Takeo, Ito, Hikaru, Inagaki, Masaki
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.08.1997
• Subjects: cdc2 kinase; CDC2 Protein Kinase - chemistry; CDC2 Protein Kinase - metabolism; cdk 5; Computer Simulation; Cyclin-Dependent Kinase 5; Cyclin-Dependent Kinases; Kinetics; molecular dynamics and molecular mechanics simulations; Oligopeptides - chemical synthesis; Oligopeptides - metabolism; peptide synthesis; Phosphorylation; Proline - chemistry; Protein Structure, Secondary; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - metabolism; Pyrrolidines - chemistry; Serine - metabolism; Substrate Specificity; Vimentin - metabolism
• ISSN: 0021-924X
• DOI: 10.1093/oxfordjournals.jbchem.a021768

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 Km 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 Km value 7- to 40-fold for cdc2 kinase. For cdk5, these substitutions led to parallel effects on the Km 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 Km 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 Vmax value for cdc2 kinase or especially for cdk5. These will aid in designing specific substrates or inhibitors for cdc2 kinase or cdk5.