MECHANISM OF ENERGY COUPLING IN THE F0F1-ATP SYNTHASE - THE UNCOUPLING MUTATION, GAMMA-M23K, DISRUPTS THE USE OF BINDING-ENERGY TO DRIVE CATALYSIS

The Escherichia coli F0F1 ATP synthase uncoupling mutation, gamma M23K , was found to increase the energy of interaction between gamma and be ta subunits which caused inefficient transmission of coupling informat ion between transport and catalysis [Al-Shawi, M. K., Ketchum, C. J., and Nakamoto, R. K. (1997) J. Biol. Chem. 272, 2300-2306]. We hypothes ized that the gamma M23K mutation, because of its effect on coupling, should alter the fundamental reactions steps that are normally modulat ed by Delta mu(H)+ via the coupling mechanism. In this paper, we addre ss this issue by studying the thermodynamics of individual catalytic s teps through the use of energy profiles to gain information regarding enzyme mechanism and the effects of the mutation. Compared to wild-typ e enzyme, the gamma M23K F-1 had significant differences of two partia l reactions: the rate constant for P-i 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 F-1 ATPase mutant enzymes and were analyzed quantitatively by linear free energy relationships [Al-Shawl, M. K., Parsonage, D., a nd Senior, A. E., (1990) J. Biol. Chern. 265, 4402-4410]. We found tha t the gamma M23K mutation prevents the proper utilization of binding e nergy to drive catalysis and blocks the enzyme in a P-i release mode. This finding is consistent with the use of energy from Delta mu(H)+ fo r increasing the affinity for P-i so that the substrate binds in a cat alytically competent manner for synthesis of ATP. These results suppor t the notion that the communication of coupling information is transmi tted through the gamma-beta interface near gamma Met23 and beta(380)DE LSEED(386) segment.