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.

Establishment and Application of a High Throughput Model for Rho Kinase Inhibitors Screening Based on Fluorescence Polarization

“…Trauger's report 18) suggested that S6-peptide is also one of selective substrates for ROCK, and conformed by Turner et al 16) The IC 50 of Y-27632 based on fluorescent S6-peptide in present study was similar to Turner's report, 16) which suggested the FP assay system is effective to determine ROCK activity. Takami et al 10) had successfully screened 69000 compounds based on SPA model, the substrate in their experiment also was S6-peptide.…”

Harvest Active Recombinant Rho Kinase from Escherichia coli

“…The classical selective substrates for ROCK are myosin light chain, 19) and myosin light chain phosphatase, 20) which regulate actin contraction. Trauger's report 21) suggested that S6-derived peptide is also one of selective substrates for ROCK, and conformed by Turner et al 12) The constants of hROCK (Table 1) and IC 50 of Y-27632 were similar to Turner's report, 12) which suggested the FP assay system is effective and feasible to determine ROCK activity. Compared with hROCK-CD, the parameters of rROCK-CD (including activity, K m , K cat , and IC 50 of Y-27632) based on the activity assay system were also similar; which indicated that the purified rROCK-CD could be used for drug screening or further activity research.…”

Pharmacological Characterization of SAR407899, a Novel Rho-Kinase Inhibitor

“…K i values for human ROCK-2 for the known ROCK inhibitors Y-27632 and fasudil were determined as 114Ϯ11 nM and 271Ϯ14 nM, respectively, in good agreement with values described previously. 31,32 Thus, Y-27632 and fasudil are Ϸ3 times and 8 times less potent than SAR407899. Like SAR407899, both compounds were found to be ATP competitive.…”

“…According to the Cheng-Prusoff equation, 32 the IC 50 value for equipotent, ATP-competitive compounds would, thus, be expected to be 4.7ϫK i for ROCK-2 or 11.0ϫK i for ROCK-1. Therefore, it can be concluded that fasudil and Y-27632, as well as SAR407899 and structurally related compounds, are not selective for both ROCK isoforms.…”