Kinetic mechanism of fully activated S6K1 protein kinase.
S6K1 is a member of the AGC subfamily of serine-threonine protein kinases, whereby catalytic activation requires dual phosphorylation of critical residues in the conserved T-loop (Thr-229) and hydrophobic motif (Thr-389). Previously, we described production of the fully activated catalytic kinase domain construct, His(6)-S6K1alphaII(DeltaAID)-T389E. Now, we report its kinetic mechanism for catalyzing phosphorylation of a model peptide substrate (Tide, RRRLSSLRA). First, two-substrate steady-state kinetics and product inhibition patterns indicated a Steady-State Ordered Bi Bi mechanism, whereby initial high affinity binding of ATP (K(d)(ATP)=5-6 microM) was followed by low affinity binding of Tide (K(d)(Tide)=180 microM), and values of K(m)(ATP)=5-6 microM and K(m)(Tide)=4-5 microM were expressed in the active ternary complex. Global curve-fitting analysis of ATP, Tide, and ADP titrations of pre-steady-state burst kinetics yielded microscopic rate constants for substrate binding, rapid chemical phosphorylation, and rate-limiting product release. Catalytic trapping experiments confirmed rate-limiting steps involving release of ADP. Pre-steady-state kinetic and catalytic trapping experiments showed osmotic pressure to increase the rate of ADP release; and direct binding experiments showed osmotic pressure to correspondingly weaken the affinity of the enzyme for both ADP and ATP, indicating a less hydrated conformational form of the free enzyme.
Keshwani
Malik M
MM
Department of Chemistry, University of Miami, Coral Gables, Florida 33124, USA.
Harris
Thomas K
TK
D000244:Adenosine Diphosphate / Q000378:metabolism; D004789:Enzyme Activation; D007700:Kinetics; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D019893:Ribosomal Protein S6 Kinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D050505:Mutant Proteins; D000244:Adenosine Diphosphate; D019893:Ribosomal Protein S6 Kinases