title: Kinetic mechanism for human Rho-Kinase II (ROCK-II).
abstract: Rho-Kinase is a serine/threonine kinase that is involved in the regulation of smooth muscle contraction and cytoskeletal reorganization of nonmuscle cells. While the signal transduction pathway in which Rho-Kinase participates has been and continues to be extensively studied, the kinetic mechanism of Rho-Kinase-catalyzed phosphorylation has not been investigated. We report here elucidation of the kinetic mechanism for Rho-Kinase by using steady-state kinetic studies. These studies used the kinase domain of human Rho-Kinase II (ROCK-II 1-534) with S6 peptide (biotin-AKRRRLSSLRA-NH(2)) as the phosphorylatable substrate. Double-reciprocal plots for two-substrate kinetic data yielded intersecting line patterns with either ATP or S6 peptide as the varied substrate, indicating that Rho-Kinase utilized a ternary complex (sequential) kinetic mechanism. Dead-end inhibition studies were used to investigate the order of binding for ATP and the peptide substrate. The ATP-competitive inhibitors AMP-PCP and Y-27632 were noncompetitive inhibitors versus S6 peptide, and the S6 peptide analogue S6-AA (acetyl-AKRRRLAALRA-NH(2)) was a competitive inhibitor versus S6 peptide and a noncompetitive inhibitor versus ATP. These results indicated a random order of binding for ATP and S6 peptide.
authors.affiliation: Department of Molecular Neuroscience, Merck Research Laboratories, 3535 General Atomics Court, San Diego, CA 92121, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Trauger
authors.forename: Fen-Fen
authors.initials: FF
authors.lastname: Lin
authors.forename: Mary S
authors.initials: MS
authors.lastname: Turner
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Stephens
authors.forename: Philip V
authors.initials: PV
authors.lastname: LoGrasso
mesh_terms: D001921:Brain / Q000201:enzymology; D015723:Gene Library; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007700:Kinetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D054460:rho-Associated Kinases
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D011994:Recombinant Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases