The MgATP-binding site on chicken gizzard myosin light chain kinase remains open and functionally competent during the calmodulin-dependent activation-inactivation cycle of the enzyme.
An ATP-like affinity labeling reagent, 5'-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA), was used to probe the MgATP-binding site of smooth muscle myosin light chain kinase from chicken gizzard (smMLCK) and its calmodulin (CaM) complex. Native smMLCK has an absolute requirement for the binding of the calcium complex of CaM for expression of its catalytic activity. FSBA reacted with smMLCK-CaM and with the CaM-free, inactive enzyme as well. Both reactions were dependent on time and FSBA concentration. Reaction was accompanied by the incorporation of covalently bound [14C]FSBA into smMLCK protein at a molar ratio of approximately 1:1 in each case. p-(Fluorosulfonyl)benzoic acid, an analogue of FSBA lacking the adenosine targeting group, did not react at a significant rate with either form of smMLCK. Reaction of CaM-free and CaM-bound smMLCK with FSBA displayed saturation kinetics. The first-order rate constants for the conversion of the reversible, noncovalent enzyme-FSBA complex to form the irreversibly inhibited, covalently modified enzyme were similar for both smMLCK and smMLCK-CaM, 0.15 and 0.07 min-1, respectively. The concentrations of FSBA yielding the half-maximal rate of inactivation, KI, were essentially identical--0.65 and 0.64 mM, respectively--for smMLCK and smMLCK-CaM. MgATP, but not MgGTP or a substrate peptide, potently inhibited reaction with FSBA. Inhibition by MgATP was competitive. The measured inhibitory constant for MgATP was essentially the same--33 versus 34 microM--for both smMLCK and smMLCK-CaM. It therefore is concluded that the MgATP-binding site on smMLCK remains accessible and recognizable as such when the enzyme becomes inactivated upon dissociation of CaM.
Kennelly
P J
PJ
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
Leng
J
J
Marchand
P
P
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D002645:Chickens; D004789:Enzyme Activation; D005895:Gizzard, Avian; D007700:Kinetics; D008024:Ligands; D009219:Myosin-Light-Chain Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000648:ultrastructure; D011487:Protein Conformation
D002147:Calmodulin; D008024:Ligands; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase