A Rotor-Stator Cross-link in the F1-ATPase Blocks the Rate-limiting Step of Rotational Catalysis

Abstract

The F 0 F 1 -ATP synthase couples the functions of H ؉ transport and ATP synthesis/hydrolysis through the efficient transmission of energy mediated by rotation of the centrally located ␥, ⑀, and c subunits. To understand the ␥ subunit role in the catalytic mechanism, we previously determined the partial rate constants and devised a minimal kinetic model for the rotational hydrolytic mode of the F 1 -ATPase enzyme that uniquely fits the pre-steady state and steady state data (Baylis Scanlon, J. A., Al-Shawi, M. K., Le, N. P., and Nakamoto, R. K. (2007) Biochemistry 46, 8785-8797). Here we directly test the model using two single cysteine mutants, ␤D380C and ␤E381C, which can be used to reversibly inhibit rotation upon formation of a cross-link with the conserved ␥Cys-87. In the pre-steady state, the ␥-␤ cross-linked enzyme at high Mg⅐ATP conditions retained the burst of hydrolysis but was not able to release P i . These data show that the rate-limiting rotation step, k ␥ , occurs after hydrolysis and before P i release. This analysis provides additional insights into how the enzyme achieves efficient coupling and implicates the ␤Glu-381 residue for proper formation of the rate-limiting transition state involving ␥ subunit rotation.

Temperature Dependence of Single Molecule Rotation of the Escherichia coli ATP Synthase F1 Sector Reveals the Importance of γ-β Subunit Interactions in the Catalytic Dwell

“…6B. The rate constants for each step are found in Scanlon et al (22,23). With the enzyme in V max conditions, we only observe the pauses at the 120°positions (32).…”

“…Pre-steady state analysis of the burst kinetics of ATP hydrolysis at nearly V max conditions demonstrated that the rate-limiting transition state occurs after the reversible hydrolysis/synthesis step and before release of phosphate (P i ) (22,23). The rate-limiting step is likely associated with a rotation step because a ␥-␤ cross-linked enzyme is still able to undergo the initial ATP hydrolysis, but the rotation-impeded enzyme is unable to release P i (23). Significantly, the kinetics of steady state hydrolysis can only be assessed when the Mg⅐ATP concentration is high enough to fill all three catalytic sites.…”

“…The only model consistent with these data is one that involves all three catalytic sites. During each 120°catalytic cycle, one site binds ATP, a different site carries out reversible hydrolysis/ synthesis, and the third site releases product P i and ADP (22,23).…”

Double-lock ratchet mechanism revealing the role of αSER-344 in F o F 1 ATP synthase

“…Because the sequence of the four active site conformations considered here is most likely β TP → β DP → β HC → β E toward direction of hydrolysis (12,25), we conclude that III TS and II TS are most likely somewhat separated in time by the conformational changes of the active site. We notice that hydrolysis of ATP takes place after an approximately 90°substep of the γ-subunit (10) and P i release also takes place following an approximately 90°γ-subunit substep (10,20). In view of our current results, it thus seems logical that, in a selected active site, ATP hydrolysis and P i release require two separate 90°γ-subunit steps or, in other words, during the 2-ms waiting dwells these occur approximately in synchrony, but in different active sites.…”

“…Considering the function of the ATP synthase, structure determinations (12)(13)(14)(15)(16), mutation studies on the α-, β-, and γ-subunits (1,(17)(18)(19)(20), single-molecule studies (6,7,10), and simulations (11,(21)(22)(23)(24)(25)(26)(27)) have contributed to a rather detailed understanding of its mode of mechanical action. However, one of several issues that need to be better characterized (1) is the mechanistic resolution of the chemical reaction steps on the way from ADP to ATP at an atomic level.…”