Metal dependence and thermodynamic characteristics of the beef heart mitochondrial adenosine triphosphatase.

A systematic study was done examining the steady state kinetics of F1-catalyzed nucleotide hydrolysis in the presence of various activating divalent metal cations. Values of Km and kcat were obtained from Lineweaver-Burk plots, and kcat/Km values were calculated. With some exceptions, kcat/Km was shown to be independent of the metal present for F1-catalyzed ATP hydrolysis, independent of the nucleotide hydrolyzed with magnesium as the metal cation present, and independent of temperature with most activating metal cations. An average value for kcat/Km of 2.62 X 10(5) M-1 S-1 is calculated as the lower limit to the second order rate constant for binding substrate to enzyme. Changes in steady state kinetic parameters with temperature were also studied. The Ki value for ADP inhibition of F1-catalyzed ATP hydrolysis with magnesium as divalent cation present was found to be temperature-independent. Plots of log kcat versus 1/T showed either abrupt breaks or straight line dependencies depending on the metal ion present. These results may indicate that different rate-limiting steps in the reaction sequence can be operative at different temperatures depending on the divalent cation present.