Kinetic and catalytic mechanisms of protein kinase A

RINALDI, J., ROSSI, S. AND MORENO, S.

Rosario, Santa Fe, Argentina

Congreso; 42 Reunion Anual de SAIB; 2006

SAIB

Like all Ser-Thr Protein Kinase, PKA is a ATP-dependent phosphotransferase that delivers a single phospohryl group from the g position of ATP to the hydroxyls of a Ser or a Thr in protein substrates. It is a heterotetramer composed of a regulatory subunit dimer (R2) and two catalytic subunits (2C).The inhibitory site (IS) of R subunit docks to the active site of C subunit. The kinetic pathway for substrate phosphorylation by PKA is composed of three fundamental events: substrate binding, the phosphoryl transfer step, and irreversible net product release. While the simple core structure is shared within the protein kinases family, the core frequently interacts with regulatory proteins or domains that either enhance or repress catalytic function.For instance, biochemical assays perfomed with synthetic peptides in PKA system have shown that the presence of the 40-residue region N-terminal to the IS enhances the activity of C subunit. To evaluate which of the reaction steps are being affected by this region we have estimated the effect of solvent viscosity on the kcat. kcat was fully sensitive to solvent viscosity using kempide as substrate, indicating the rate-limiting step is the net product release; while when using the peptide that includes the N-terminal to the IS region the effect is intermediate, implying that phosphoryl transfer and net product release are both rate-determining.