NUCLEOTIDE BINDING AND STEADY-STATE KINETICS OF YEAST HEXOKINASE AND BEEF HEART MITOCHONDRIAL ADENOSINE TRIPHOSPHATASE

Nucleotide binding to hexokinase is studied using fluorometric and kinetic techniques. Dissociation constants for (epsilon)ATP and (epsilon)ADP with hexokinase are obtained from fluorometric measurements and compared with similar constants obtained kinetically. Other selected nucleoside triphosphates are used as phosphate donors in the hexokinase reaction to obtain their kinetic constants. Evidence is presented to support the hypothesis that the K(,m)\u27s are primarily dissociation constants in a random bi-bi mechanism. The roles of tyrosine and divalent metals in the catalytic mechanism of nucleoside triphosphate hydrolysis by beef heart mitochondrial ATPase are explored. Data are presented showing that the phenolic group of an active-site tyrosine must be protonated during catalysis. Sequential modifications of arginine and tyrosine suggest a close proximity of these amino acids in the active site of F(,1). Kinetic constants are obtained for the hydrolyses of several nucleoside triphosphates by F(,1) in the presence of several divalent metals. Arrhenius plots indicate the existence of a different rate-limiting step at temperatures above 20(DEGREES)C compared to lower temperatures. The rate-limiting step for MgATP and MgITP hydrolyses by F(,1) below 20(DEGREES)C is deuterium-sensitive. Above 20(DEGREES)C, the rate-limiting step is deuterium-sensitive for MgITP hydrolysis and deuterium-insensitive for MgATP hydrolysis. The V/K value for ATP hydrolysis by F(,1) is unaffected by changes in temperature, deuterium concentration, or the divalent cation present. These results suggest that V/K is the binding rate constant of ATP to F(,1) with a value of 2.62 x 10(\u275) sec(\u27-1) M(\u27-1). Evidence is presented which supports the hypothesis that the pk(,a) of a metalbound water contributes to the activity of the enzyme in nucleoside triphosphate hydrolysis and synthesis. The magnitude of the deuterium effect on ATP hydrolysis by F(,1) increases as the metal ion involved is changed from Ca to Mg to Ni. Nitration of tyrosine affects the enzymatic ATPase reaction in the order Ca \u3e Mg \u3e Ni. These data are used to support a mechanism for the migration of the metal along the nucleotide phosphate chain during nucleoside triphosphate hydrolysis and synthesis by F(,1). This mechanism is incorporated into a complete scheme for the catalytic reactions of F(,1)