Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast.
Cak1p, the Cyclin-dependent kinase-activating kinase from budding yeast, is an unusual protein kinase that lacks many of the highly conserved motifs observed among members of the protein kinase superfamily. Cak1p phosphorylates and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this paper, we explore the kinetics of CDK phosphorylation by Cak1p, and we examine the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reasonable affinities, exhibiting K(d) values of 7.2 and 0.6 microm, respectively. Interestingly, these values are approximately the same as the K(M) values, indicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Cak1p is a slow enzyme, with a catalytic rate of only 4.3 min(-)(1). The absence of a burst phase indicates that product release is not rate-limiting. This result, and a solvent isotope effect, suggests that a catalytic step is rate-limiting.
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06520-8114, USA.
D A
DA
Enke
P
P
Kaldis
M J
MJ
Solomon
D000255:Adenosine Triphosphate / Q000494:pharmacology; D042846:CDC2-CDC28 Kinases; D002384:Catalysis; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004795:Enzyme Stability; D007700:Kinetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D004718:Saccharomycetales / Q000201:enzymology*; D000095043:Cyclin-Dependent Kinase-Activating Kinase
D000255:Adenosine Triphosphate; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D000095043:Cyclin-Dependent Kinase-Activating Kinase