Activation mechanism of rabbit skeletal muscle myosin light chain kinase. 5'-p-fluorosulfonylbenzoyl adenosine as a probe of the MgATP-binding site of the calmodulin-bound and calmodulin-free enzyme.
5'-p-fluorosulfonylbenzoyl adenosine (FSBA), an ATP-like affinity labelling reagent, reacted with rabbit skeletal muscle myosin light chain kinase (skMLCK) and its calmodulin complex in a site-specific manner. Reaction was dependent upon the presence of the adenosine moiety of FSBA, saturated with increasing FSBA, was inhibited by MgATP, and was accompanied by stoichiometric incorporation of [14C]FSBA. The kinetic constants describing the reaction were similar for skMLCK and its calmodulin complex: k3 = -0.040 min-1 and -0.038 min-1, and Ki = 0.18 mM and 0.40 mM, respectively. It is concluded that the MgATP-binding site on skMLCK remains accessible at all times and maintains a near constant conformation.
Kennelly
P J
PJ
Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
Colburn
J C
JC
Lorenzen
J
J
Edelman
A M
AM
Stull
J T
JT
Krebs
E G
EG
D000241:Adenosine / Q000031:analogs & derivatives; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002147:Calmodulin / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits
D000345:Affinity Labels; D002147:Calmodulin; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D000241:Adenosine