ATP Hydrolysis in the βTP and βDP Catalytic Sites of F1-ATPase

Abstract

The enzyme F1-adenosine triphosphatase (ATPase) is a molecular motor that converts the chemical energy stored in the molecule adenosine triphosphate (ATP) into mechanical rotation of its gamma-subunit. During steady-state catalysis, the three catalytic sites of F1 operate in a cooperative fashion such that at every instant each site is in a different conformation corresponding to a different stage along the catalytic cycle. Notwithstanding a large amount of biochemical and, recently, structural data, we still lack an understanding of how ATP hydrolysis in F1 is coupled to mechanical motion and how the catalytic sites achieve cooperativity during rotatory catalysis. In this publication, we report combined quantum mechanical/molecular mechanical simulations of ATP hydrolysis in the betaTP and betaDP catalytic sites of F1-ATPase. Our simulations reveal a dramatic change in the reaction energetics from strongly endothermic in betaTP to approximately equienergetic in betaDP. The simulations identify the responsible protein residues, the arginine finger alphaR373 being the most important one. Similar to our earlier study of betaTP, we find a multicenter proton relay mechanism to be the energetically most favorable hydrolysis pathway. The results elucidate how cooperativity between catalytic sites might be achieved by this remarkable molecular motor.

A phosphoryl transfer intermediate in the GTPase reaction of Ras in complex with its GTPase-activating protein

“…An attractive explanation for the production of this species is the extensively discussed possibility of transfer (direct or indirect) of a proton from the attacking water molecule to an oxygen of the ␥-phosphate group (9,16,17,(35)(36)(37), a mechanism that has been called substrate-assisted catalysis (16). Such proton transfer can be direct or mediated via water molecules, as has been discussed for Ras and the F1 ATPase reaction (4,38). After attack of the generated hydroxyl ion on phosphorus and cleavage of the ␤,␥-bond, H 2 PO 4 Ϫ would be generated as the initial product.…”

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

“…Single-molecule studies showed that the 120°rotational steps can be further divided into approximately 90°and 30°substeps during hydrolysis (7, 10). A roughly 2-ms waiting time is observed between these substeps, most likely due to two separate 1-ms steps, one corresponding to the hydrolysis or synthesis step, the other to P i release or binding (10).Considering the function of the ATP synthase, structure determinations (12-16), mutation studies on the α-, β-, and γ-subunits (1, 17-20), 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.…”

“…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.…”

“…In order to understand the effects of the active site conformation along the reaction, models using both the β DP and β TP active sites as well as conformational changes between β HC , β DP , and β TP active sites were considered. This approach was inspired by the pioneering studies by Dittrich et al (21,22) and Štrajbl et al (27) on ATPase reaction mechanism. In the former, the authors used a lower level of theory on a 70 atom QM part and a crystal structure with ADP and not ATP in the active sites (15).…”

Operation mechanism of FoF1‐adenosine triphosphate synthase revealed by its structure and dynamics

“…This positional shift of the a-subunit makes the well-conserved arginine residue in the a-subunit, known as ''Arg-finger,'' very close to the c-phosphate of ATP in the range of a few Ås (4). Although some biochemical experiments and simulations have indicated that the Arg-finger accelerates the ATP hydrolysis rate by stabilizing the transition state (5,6), it is dispensable for the rotary catalysis (7). The ATP hydrolysis step is not thought to be a major torque-generating step, because accompanying conformational change appears to be too small to induce rotation of the csubunit.…”