J. Biol. Chem., Vol. 266, Issue 13, 8073-8078, 05, 1991

Alteration of apparent negative cooperativity of ATPase activity by alpha-subunit glutamine 173 mutation in yeast mitochondrial F1. Correlation with impaired nucleotide interaction at a regulatory site

JM Jault, A Di Pietro, P Falson and DC Gautheron
Laboratoire de Biologie et Technologie des Membranes et des Systemes Integres, UMR 24 du Centre National de la Recherche Scientifique, Universite Claude Bernard de Lyon, Villeurbanne, France.

The first described alpha-subunit mutation of yeast mitochondrial F1 has been recently identified as a single Gln173----Leu substitution in a strongly conserved sequence (Falson, P., Maffey, L., Conrath, K., and Boutry, M. (1991) J. Biol. Chem. 266, 287-293). This mutation is shown here to greatly modify the biphasic pattern of ATPase activity as a function of pH: (i) the shoulder observed at acidic pH is significantly increased; (ii) the main peak, at alkaline pH, is markedly lowered; (iii) the optimal pH is shifted from 8.8 to 7.7. The mutation lowers both apparent negative cooperativity and sensitivity to azide inhibition which concomitantly increase when the assay pH decreases. Azide partial inhibition produces apparent negative cooperativity which can be further abolished by bicarbonate. The mutation increases both activation energies determined from biphasic Arrhenius plots. The mutation decreases the inactivation rate by 5'-p- fluorosulfonylbenzoyladenosine and abolishes the protection by nucleotide binding at the adenine-specific regulatory site. On the contrary, it does not modify the reactivity of 5'-p- fluorosulfonylbenzoylguanosine at the less-selective catalytic site. In addition, partial inactivation by 5'-p-fluorosulfonylbenzoyladenosine, as opposed to 5'-p-fluorosulfonylbenzoylguanosine, produces apparent negative cooperativity under conditions where unmodified-enzyme kinetics are noncooperative. The results show that alpha-Gln173 participates in nucleotide interaction at a regulatory site which controls the negative cooperativity of F1-ATPase activity.
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