The Escherichia coli F0F1 gamma M23K uncoupling mutant has a higher K-0.5 for P-i. Transition state analysis of this mutant and others reveals that synthesis and hydrolysis utilize the same kinetic pathway

The Escherichia coli F0F1 ATP synthase uncoupling mutation, gamma M23K, was found to increase the energy of interaction between gamma and beta subunits, prevent the proper utilization of binding energy to drive catalysis, and block the enzyme in a P-i release mode. In this paper, the effects of this mutation on substrate binding in cooperative ATP synthesis are assessed. Activation of ATP synthesis by ADP and P-i was determined for the gamma M23K F0F1. The K-0.5 for ADP was not affected, but K-0.5 for P-i was approximately 7-fold higher even though the apparent V-max was close to the wild-type level. Wild-type enzyme had a turnover number of 82 s(-1) at pH 7.5 and 30 degrees C. During oxidative phosphorylation, the apparent dissociation constant (K-1) for ATP was not affected and was 5-6 mM for both wild-type and gamma M23K enzymes. Thus, the apparent binding affinity for ATP in the presence of Delta mu(H)+ was lowered by 7 orders of magnitude from the affinity measured at the high-affinity catalytic site. Arrhenius analysis of ATP synthesis for the gamma M23K F0F1 revealed that, like those of ATP hydrolysis, the transition state Delta H double dagger was much more positive and T Delta S double dagger. was much less negative, adding up to little change in Delta G double dagger. These results suggested that ATP synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. Analysis of the transition state structures using isokinetic plots demonstrate that ATP hydrolysis and synthesis utilize the same kinetic pathway. Incorporating this information into a model for rotational catalysis suggests that at saturating substrate concentrations, the rate-limiting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.