Nonfixed relationship of the Michaelis constant and maximum velocity with their corresponding rate constants.
The Michaelis constant (K(m)) and V(mas) (E0k(cat)) values for two mutant sets of enzymes were studied from the viewpoint of their definition in a rapid equilibrium reaction model and in a steady state reaction model. The "AMP set enzyme" had a mutation at the AMP-binding site (Y95F, V67I, and V67I/L76V), and the "ATP set enzyme" had a mutation at a possible ATP-binding region (Y32F, Y34F, and Y32A/Y34A). Reaction rate constants obtained using steady state model analysis explained discrepancies found by the rapid equilibrium model analysis. (i) The unchanged number of bound AMPs for Y95F and the wild type despite the markedly increased K(m) values for AMP of the AMP set of enzymes was explained by alteration of the rate constants of the AMP step (k(+2), k(-2)) to retain the ratio k(+2)/k(-2). (ii) A 100 times weakened selectivity of ATP for Y34F in contrast to no marked changes in K(m) values for both ATP and AMP for the ATP set of enzymes was explained by the alteration of the rate constants of the ATP steps. A similar alteration of the K(m) and k(cat) values of these enzymes resulted from distinctive alterations of their rate constants. The pattern of alteration was highly suggestive. The most interesting finding was that the rate constants that decided the K(m) and k(cat) values were replaced by the mutation, and the simple relationships between K(m), k(cat), and the rate constants of K(m)1 = k(+1)/k(-1) and k(cat) = k(f) were not valid. The nature of the K(m) and k(cat) alterations was discussed.
Hibino
Takeshi
T
Laboratory of Biophysical Chemistry, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000263:Adenylate Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008962:Models, Theoretical; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013552:Swine
D007527:Isoenzymes; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000263:Adenylate Kinase