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 be ta 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 affecte d, but K-0.5 for P-i was approximately 7-fold higher even though the a pparent 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 oxidat ive phosphorylation, the apparent dissociation constant (K-1) for ATP was not affected and was 5-6 mM for both wild-type and gamma M23K enzy mes. Thus, the apparent binding affinity for ATP in the presence of De lta mu(H)+ was lowered by 7 orders of magnitude from the affinity meas ured at the high-affinity catalytic site. Arrhenius analysis of ATP sy nthesis for the gamma M23K F0F1 revealed that, like those of ATP hydro lysis, the transition state Delta H double dagger was much more positi ve and T Delta S double dagger. was much less negative, adding up to l ittle change in Delta G double dagger. These results suggested that AT P synthesis is inefficient because of an extra bond between gamma and beta subunits which must be broken to achieve the transition state. An alysis of the transition state structures using isokinetic plots demon strate that ATP hydrolysis and synthesis utilize the same kinetic path way. Incorporating this information into a model for rotational cataly sis suggests that at saturating substrate concentrations, the rate-lim iting step for hydrolysis and synthesis is the rotational power stroke where each of the beta subunits changes conformation and affinity for nucleotide.