Kinetic studies on rabbit-muscle myokinase

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Abstract
A phosphorylated enzyme intermediate could not be detected when purified myokinase was incubated with adenosine triphosphate (ATP) and AT[beta]gamma32P, comparable concentrations of enzyme and substrate being used, and the enzyme was separated from the substrate by dialysis or zone electrophoresis. The Michaelis constant (Km) and enzyme-inhibitor dissociation constant (Ki) values for the substrate ATP are 0.3 m[image] and 0.32 m[image] respectively. For adenosine diphosphate (ADP), Km= Ki = 1.58 m[image]. For adenosine monophosphate (AMP), the Km and Ki values are 0.5 m[image]. Kinetic studies showed that ATP and AMP inhibited the forward reaction competitively. A one-site theory for the mechanism of action of myokinase is proposed and discussed, evidence being brought to show that there is no demonstrable second site on the enzyme for any of the substrates. A theoretical treatment is given for the detection of secondary association constants. p-Chloromercuribenzoate inhibits myokinase non-competitively (Ki = 0.015 [mu][image]) and adenosine 5[image]-monosulfate inhibits myokinase competitively (Ki = 18.6 [mu][image]). Other nucleotides tested did not show detectable inhibition of myokinase, and aniline and benzoate were without effect. Apparently the amino and phosphate groups as well as the adenine ring are specifically required for enzyme activity, although the sulfate group may be substituted for phosphate for purposes of contact with the active center. A trace of nucleoside monophosphate kinase activity was detected in the myokinase preparations. Myokinase is activated by Mg++. Optimum activation occurs when the Mg++ concentration is of the same order as the substrate concentration (0.01 [image]). The enzyme is inhibited 56-62% by 0.02 [image] NaF.