Temperature-induced transitions in the conformation of intermediates in the hydrolytic cycle of myosin.
The conformations of the transitory intermediates of the myosin ATPase occurring during the hydrolytic cycle, enzyme without ligand, enzyme-substrate complex and two different forms of enzyme-product complex, have been characterized in terms of numbers and classes of reactive thiol groups based on incorporation of radioactively labeled alkylation reagent. The techniques employed allowed this to be done under steady-state conditions in the presence of high ligand concentrations on intact myosin from rabbit fast skeletal muscles at low ionic strength where the protein is in the gel state as it is in muscle. The binding of a divalent cation (Mg2+ or Ca2+) nucleotide complex exposes thiol-1 as well as thiol-2 groups. The long-lived ATPase intermediate occurring at temperatures above 10 degrees C adopts the same conformation with Mg2+ and Ca2+ ions. This intermediate does not protect the thiol-1 and thiol-2 groups but exposes a number of thiol-3 groups which seem to be located distant from the active site. The conformation of the intermediate prevailing in the presence of ATP changes with lowering temperature below 10 degrees C and is identical with that found in the presence of ADP at 0 degree C indicating a change in the rate-limiting step of the hydrolytic cycle. In the absence of divalent cations no such temperature-dependent change in conformation was observed. Evaluation of the activation entropies shows that the structure of the long-lived intermediate occurring above 10 degrees C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20 degrees C. In the case of the monovalent-cation-stimulated ATPase a constant activation energy of around 70 kJ/mol, typical of many enzyme reactions, was found over the entire temperature range from 0--35 degrees C.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D004789:Enzyme Activation / Q000187:drug effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009132:Muscles / Q000201:enzymology; D009218:Myosins / Q000378:metabolism; D009994:Osmolar Concentration; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013696:Temperature
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D009218:Myosins; D008274:Magnesium; D005033:Ethylmaleimide; D011188:Potassium