Evaluation of the catalytic mechanism of the p21-activated protein kinase PAK2.
The p21-activated kinases (PAKs) play important roles in diverse cellular processes. In the present study, we provide an in-depth kinetic analysis of one of the PAK family members, PAK2, in phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate derived from LIM kinase, LIMKtide. Steady-state kinetic analysis of the initial reaction velocity of PAK2 phosphorylation of MBP is consistent with both randomly and compulsorily ordered mechanisms. Further kinetic studies carried out in various concentrations of sucrose revealed that solvent viscosities had no effect on k(cat)/K(m) for either ATP or MBP while k(cat) was highly sensitive to solvent viscosity, indicating that the enzymatic phosphorylation by PAK2 can be best interpreted by a rapid-equilibrium random bi-bi reaction model, and k(cat) is partially limited by both phosphoryl group transfer (31 s(-)(1)) and the product release (19 s(-)(1)). In the phosphorylation of LIMKtide, both k(cat) and k(cat)/K(m) were insensitive to solvent viscosity, and the product release (86 s(-)(1)) was much faster than the phosphoryl group transfer step (19 s(-)(1)). These studies suggest that the release of phospho-MBP product is likely partially rate determining for the PAK2-catalyzed reaction since the dissociation rate of products from the PAK2 active site for LIMKtide phosphorylation differs from that of MBP significantly. Such a mechanism is in contrast to the previously established kinetics for the phosphorylation of peptide substrates by cAMP-dependent kinase, in which this process is limited by the release of ADP but not the phospho-peptide product. These results complement previous structure-function studies of PAKs and provide important insight for mechanistic interpretation of the kinase functions.
Wu
Hao
H
National Laboratory of Biomacromolecules, Center for Molecular Biology, Institute of Biophysics, Academia Sinica, Beijing 100101, People's Republic of China.
Zheng
Yi
Y
Wang
Zhi-Xin
ZX
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D054461:Lim Kinases; D008956:Models, Chemical; D008969:Molecular Sequence Data; D004676:Myelin Basic Protein / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013379:Substrate Specificity; D014783:Viscosity; D054462:p21-Activated Kinases
D004676:Myelin Basic Protein; D011993:Recombinant Fusion Proteins; D012997:Solvents; D000255:Adenosine Triphosphate; D011494:Protein Kinases; C515892:LIMK1 protein, human; D054461:Lim Kinases; C515907:PAK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054462:p21-Activated Kinases