Please use this identifier to cite or link to this item: https://doi.org/10.1021/bi700421h
DC FieldValue
dc.titleCyclic-AMP and pseudosubstrate effects on type-I A-kinase regulatory and catalytic subunit binding kinetics
dc.contributor.authorAnand, G.
dc.contributor.authorTaylor, S.S.
dc.contributor.authorJohnson, D.A.
dc.date.accessioned2014-10-27T08:25:20Z
dc.date.available2014-10-27T08:25:20Z
dc.date.issued2007-08-14
dc.identifier.citationAnand, 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
dc.identifier.issn00062960
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/100391
dc.description.abstractTo 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.
dc.description.urihttp://libproxy1.nus.edu.sg/login?url=http://dx.doi.org/10.1021/bi700421h
dc.sourceScopus
dc.typeArticle
dc.contributor.departmentBIOLOGICAL SCIENCES
dc.description.doi10.1021/bi700421h
dc.description.sourcetitleBiochemistry
dc.description.volume46
dc.description.issue32
dc.description.page9283-9291
dc.description.codenBICHA
dc.identifier.isiut000248728100013
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