F1-ATPase mutations in Escherichia coli that changed the strength of hydrogen bonds between the α and β subunits in a location that links the catalytic site to the interface between the β catch loop and the γ subunit were examined. Loss of the ability to form the hydrogen bonds involving αS337, βD301, and αD335 lowered the K cat of ATPase and decreased its susceptibility to Mg 2+-ADP-AlFn inhibition, while mutations that maintain or strengthen these bonds increased the susceptibility to Mg2+-ADP- AlFn inhibition and lowered the Kcat of ATPase. These data suggest that hydrogen bonds connecting αS337 to βD301 and βR323 and connecting αD335 to αS337 are important to transition state stabilization and catalytic function that may result from the proper alignment of catalytic site residues βR182 and αR376 through the VISIT sequence (α344-348). Mutations βD301E, βR323K, and αR282Q changed the rate-limiting step of the reaction as determined by an isokinetic plot. Hydrophobic mutations of βR323 decreased the susceptibility to Mg 2+-ADP-AlFn inhibition and lowered the number of interactions required in the rate-limiting step yet did not affect the K cat of ATPase, suggesting that βR323 is important to transition state formation. The decreased rate of ATP synthase-dependent growth and decreased level of lactate-dependent quenching observed with αD335, βD301, and αE283 mutations suggest that these residues may be important to the formation of an alternative set of hydrogen bonds at the interface of the α and β subunits that permits the release of intersubunit bonds upon the binding of ATP, allowing γ rotation in the escapement mechanism.
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