Dependence of the activity of beef heart mitochondrial adenosinetriphosphatase on the properties of the catalytic metal ion.
Several divalent metal ions were used as kinetic probes of the beef heart mitochondrial adenosinetriphosphatase (F1) under a variety of conditions, and the relationship between the properties of the catalytic metal ion and the catalytic activity of the enzyme was examined. Vmax for ATP hydrolysis was largest when metal ions characterized by intermediate values of acidity of coordinated water molecules (pKa) and metal-nucleotide stability constants (Kstab) were present. As temperature increased, the peak of Vmax vs. pKa (or Kstab) shifted to lower initial values of pKa or Kstab. The solvent deuterium isotope effect on Vmax (DV) was normal and largest when the metal ion present during F1-catalyzed ATP hydrolysis was most acidic and the metal nucleotide stability constant was large. When an active site tyrosine on F1 was nitrated, Vmax was most affected when the metal ion present was least acidic and the metal nucleotide stability constant was small. The isotope effect on V/K (DV/K) was normal, small, and apparently independent of the metal ion present. ADP inhibition of F1-catalyzed ATP hydrolysis is competitive, and the Ki is independent of the metal ion present. The degree of Pi inhibition of F1 is dependent on the metal ion present. The inhibition by Pi is competitive at low temperature and becomes noncompetitive as temperature increases. These and previous results support a mechanism whereby a water molecule coordinated to the metal ion of an enzyme-bound gamma-monodentate metal-ATP complex is deprotonated to begin a series of events whereby a beta,gamma-bidentate metal-ATP complex is produced. Upon hydrolysis, the bond between the metal ion and the beta-phosphate of ADP in the Pi-metal-ADP complex is broken before products (ADP and metal-Pi) are released.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002413:Cations, Divalent; D002417:Cattle; D007700:Kinetics; D008929:Mitochondria, Heart / Q000201:enzymology*; D006180:Proton-Translocating ATPases / Q000302:isolation & purification / Q000378:metabolism*; D013816:Thermodynamics
D002413:Cations, Divalent; D000244:Adenosine Diphosphate; D006180:Proton-Translocating ATPases