Unisite catalysis without rotation of the γ-ε domain in Escherichia coli F1-ATPase

Unisite [gamma-P-32]ATP hydrolysis was studied in ECF1 from the mutant beta E381C after generating a single disulfide bond between beta and gamma subunits to prevent the rotation of the gamma/epsilon domain. The single beta-gamma cross-link was obtained by removal of the delta subunit from F-1 and then treating with CuCl2 as described previously (Aggeler, R,, Haughton, M, A., and Capaldi, R, k (1996) J. Biol, Chem. 270, 9185-9191), The mutant enzyme, beta E381C, had an increased overall rate of unisite hydrolysis of [gamma-P-32]ATP compared with the wild type ECF1 due to increases in the rate of ATP binding (k(+1)), P-i release (k(+3)), and ADP release (K+4). Release of bound substrate ([gamma-P-32]ATP) was also increased in the beta E381C mutant. Cross-linking between Cys-381 and the intrinsic Cys-87 of gamma caused a further increase in the rate of unisite catalysis, mainly by additional effects on nucleotide binding in the high affinity catalytic site (k(+1) and k(+4)), In delta-subunit-free ECF1 from wild type or beta E381C F-1, addition of an excess of ATP accelerated unisite catalysis. After cross-linking, unisite catalysis of beta E381C was not enhanced by the cold chase. The covalent linkage of gamma to beta increased the rate of unisite catalysis to that obtained by cold chase of ATP of the noncross-linked enzyme, It is concluded that the conversion of Glu-381 of beta to Cys induces an activated conformation of the high affinity catalytic site with low affinity for substrate and products. This state is stabilized by cross-linking the Cys at beta 381 to Cys-87 of gamma, We infer from the data that rotation of the gamma/epsilon rotor in ECF1 is not linked to unisite hydrolysis of ATP at the high affinity catalytic site but to ATP binding to a second or third catalytic site on the enzyme.