Pre-steady-state kinetic analysis of cAMP-dependent protein kinase using rapid quench flow techniques.

摘要: The phosphorylation of a peptide substrate by the catalytic subunit cAMP-dependent protein kinase was monitored over short time periods (2−1000 ms) using rapid quench flow mixing device and radioactive assay. production phosphokemptide [LRRAS(P)LG] as function is characterized “burst” phase (250 s-1) followed slower, linear (L/[E]t = 21 at 100 μM Kemptide. amplitude this varies linearly with enzyme concentration represents approximately 100% total concentration, indicating that not due to product inhibition. observed rate constants for phases vary hyperbolically concentration. From these dependencies, maximum constant 500 ± 60 s-1 Km Kd Kemptide 4.9 1.4 200 were determined. kcat data extracted from portion transients are indist...