On the rate of F1‐ATPase turnover during ATP hydrolysis by the single catalytic site Evidence that hydrolysis with a slow rate of product release does not occur at the alternating active site

Abstract

Under conditions of molar excess of enzyme, isolated F₁‐ATPase from beef heart mitochondria catalyses ATP hydrolysis biphasically. The rate constants for product release are ∼10⁻¹ and 10⁻⁴−10⁻³ s⁻¹, respectively. The slow phase of ATP hydrolysis is insensitive to EDTA. [γ‐³²P]ATP splitting in the slow phase cannot be chased from the enzyme during several catalytic turnovers. It follows from these results that the slow single‐site hydrolysis of ATP (k _cat∼10⁻⁴s⁻¹), initially observed by Grubmeyer et al. [(1982) J. Biol. Chem. 257, 12092–12100], is not carried out by the normal catalytic site. In contrast, the phase of rapid ATP hydrolysis (k _cat∼10⁻¹s⁻¹) is completely prevented by EDTA and is believed to be the normal function of the normal catalytic site of F₁‐ATPase.