Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk.
The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosphorylation of a substrate peptide, generated from a random library, occurs in 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 result of product inhibition. Analysis of the burst rate as a function of substrate concentration indicates that the phosphoryl transfer step is fast (k3 > or = 140 s(-1) and highly favorable (k3/k-3 > or = 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 kcat. Since the substrate dissociation constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma-phosphate of ATP to the substrate and rapidly releases the phosphoproduct. Overall rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event in the catalytic cycle is coupled with slow conformational changes.
Shaffer
J
J
Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0506, USA.
Sun
G
G
Adams
J A
JA
D000227:Adenine Nucleotides / Q000378:metabolism*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000081247:CSK Tyrosine-Protein Kinase; D017022:Flow Injection Analysis; D007700:Kinetics; D008956:Models, Chemical; D009842:Oligopeptides / Q000378:metabolism; D011487:Protein Conformation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D014783:Viscosity; D019061:src-Family Kinases
D000227:Adenine Nucleotides; D009842:Oligopeptides; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases