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 o bserved among members of the protein kinase superfamily, Cak1p phosphorylat es and activates Cdc28p, the major cyclin-dependent kinase (CDK) in yeast, and is thereby required for passage through the yeast cell cycle. In this p aper, me explore the kinetics of CDK phosphorylation by Cak1p, and me exami ne the role of the catalytic step in the reaction mechanism. Cak1p proceeds by a sequential reaction mechanism, binding to both ATP and CDK2 with reas onable affinities, exhibiting Ii, values of 7.2 and 0.6 muM, respectively. Interestingly, these values are approximately the same as the K-M values, i ndicating that the binding of substrates is fast with respect to catalysis and that the most likely reaction mechanism is rapid equilibrium random. Ca k1p is a slow enzyme, with a catalytic rate of only 4.3 min(-1). The absenc e of a burst phase indicates that product release is not rate-limiting. Thi s result, and a solvent isotope effect, suggests that a catalytic step is r ate-limiting.