Cyclic-AMP and pseudosubstrate effects on type-I A-kinase regulatory and catalytic subunit binding kinetics.
To better understand the molecular mechanism of cAMP-induced and substrate-enhanced activation of type-I A-kinase, we measured the kinetics of A-kinase regulatory subunit interactions using a stopped-flow spectrofluorometric method. Specifically, we conjugated fluorescein maleimide (FM) to two separate single cysteine-substituted and truncated mutants of the type Ialpha regulatory subunit of A-kinase, RIalpha (91-244). One site of cysteine substitution and conjugation was at R92 and the other at R239. Although the emission from both conjugates changed with catalytic subunit binding, only the FM-R92C conjugate yielded unambiguous results in the presence of cAMP and was therefore used to assess whether a pseudosubstrate perturbed the rate of holoenzyme dissociation. We found that cAMP selectively accelerates the rate of dissociation of the RIalpha (91-244):C-subunit complex approximately 700-fold, resulting in an equilibrium dissociation constant of 130 nM. Furthermore, excess amounts of the pseudosubstrate inhibitor, PKI(5-24), had no effect on the rate of RIalpha (91-244):C-subunit complex dissociation. The results indicate that the limited ability of cAMP to induce holoenzyme dissociation reflects a greatly reduced but still significant regulatory catalytic subunit affinity in the presence of cAMP. Moreover, the ability of the substrate to facilitate cAMP-induced dissociation results from the mass action effect of excess substrate and not from direct substrate binding to holoenzyme.
Anand
Ganesh
G
Department of Chemistry/Biochemistry, Howard Hughes Medical Institute, University of California, San Diego, La Jolla, California 92037, USA.
Taylor
Susan S
SS
Johnson
David A
DA
D019943:Amino Acid Substitution / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D000242:Cyclic AMP / Q000737:chemistry*; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding / Q000235:genetics; D021122:Protein Subunits / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D054753:Cyclic AMP-Dependent Protein Kinase RIbeta Subunit; D021122:Protein Subunits; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases