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|Title:||Cyclic-AMP and pseudosubstrate effects on type-I A-kinase regulatory and catalytic subunit binding kinetics|
|Authors:||Anand, G. |
|Citation:||Anand, G., Taylor, S.S., Johnson, D.A. (2007-08-14). Cyclic-AMP and pseudosubstrate effects on type-I A-kinase regulatory and catalytic subunit binding kinetics. Biochemistry 46 (32) : 9283-9291. ScholarBank@NUS Repository. https://doi.org/10.1021/bi700421h|
|Abstract:||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 Iα regulatory subunit of A-kinase, RIα (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 RIα (91-244):C-subunit complex ∼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 RIα (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. © 2007 American Chemical Society.|
|Appears in Collections:||Staff Publications|
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