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

• Published in: FEBS letters 1987-09, Vol.222 (1), p.32-36
• Main Authors: Milgrom, Ya.M., Murataliev, M.B.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Animals; Binding Sites; Biological and medical sciences; Cattle; Edetic Acid - pharmacology; Enzymes and enzyme inhibitors; F1-ATPase; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Mitochondria, Heart - enzymology; Phosphorus Radioisotopes; Proton-Translocating ATPases - metabolism; Single-site catalysis; Turnover rate
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(87)80186-2

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.