Mechanism of energy coupling in the FOF1-ATP synthase: the uncoupling mutation, gammaM23K, disrupts the use of binding energy to drive catalysis.

The Escherichia coli FOF1 ATP synthase uncoupling mutation, gammaM23K, was found to increase the energy of interaction between gamma and beta subunits which caused inefficient transmission of coupling information between transport and catalysis [Al-Shawi, M. K. , Ketchum, C. J., and Nakamoto, R. K. (1997) J. Biol. Chem. 272, 2300-2306]. We hypothesized that the gammaM23K mutation, because of its effect on coupling, should alter the fundamental reactions steps that are normally modulated by DeltamuH+ via the coupling mechanism. In this paper, we address this issue by studying the thermodynamics of individual catalytic steps through the use of energy profiles to gain information regarding enzyme mechanism and the effects of the mutation. Compared to wild-type enzyme, the gammaM23K F1 had significant differences of two partial reactions: the rate constant for Pi release was 49-fold faster and the rate constant for ATP release was 8.4-fold faster than wild-type. These rate constants were considered together with characteristics of a group of F1 ATPase mutant enzymes and were analyzed quantitatively by linear free energy relationships [Al-Shawi, M. K., Parsonage, D., and Senior, A. E., (1990) J. Biol. Chem. 265, 4402-4410]. We found that the gammaM23K mutation prevents the proper utilization of binding energy to drive catalysis and blocks the enzyme in a Pi release mode. This finding is consistent with the use of energy from DeltamuH+ for increasing the affinity for Pi so that the substrate binds in a catalytically competent manner for synthesis of ATP. These results support the notion that the communication of coupling information is transmitted through the gamma-beta interface near gammaMet23 and beta380DELSEED386 segment.