Nucleotide release and associated conformational changes regulate functionin the COOH-terminal Src kinase, Csk

The COOH-terminal Src kinase (Csk) regulates a broad array of cellular proc esses via the specific phosphorylation and downregulation of Src family pro tein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not bee n investigated. To understand active-site phenomena, pre-steady-state and t ransient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosph orylation of a substrate peptide, generated from a random library, occurs i n two kinetic phases: a rapid, exponential "burst" phase followed by a slow , linear phase. The amplitude of the burst phase increases as a function of enzyme concentration, indicating that the biphasic kinetics are not the re sult of product inhibition. Analysis of the burst rate as a function of sub strate concentration indicates that the phosphoryl transfer step is fast (k (3) greater than or equal to 140 s(-1)) and highly favorable (k(3)/k(-3) gr eater than or equal to 6). The apparent dissociation rate constant for ADP (0.6 s(-1)), measured using stopped-flow kinetic methods and a fluorescent trapping agent, mant-ATP, is close to k(cat). Since the substrate dissociat ion constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma -phos phate of ATP to the substrate and rapidly releases the phosphoproduct. Over all rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event i n the catalytic cycle is coupled with slow conformational changes.