The Role of Amino Acid Residues Located at the Catalytic Site in the Rotation of Enterococcus Hirae V1-ATPase

Abstract

V-ATPases are rotary molecular motors that generally function as proton pumps. We have recently solved the crystal structures of the V1 moiety of Enterococcus hirae V-ATPase (EhV1), providing the first high-resolution molecular structure of the V1 moiety. Additionally, we have also verified the rotary catalysis of EhV1 by using single-molecule high-speed imaging and have analyzed the properties of the rotary motion in detail. Here, to understand the role of amino acid residues located at the catalytic site on the ATP hydrolysis of EhV1, we analyzed the rotation of the mutants of EhV1. We observed the rotation of the mutants, A(F506E) (an amino acid residue which interacts with adenine ring of ATP) and B(R350K) (the arginine finger which interacts withβ- and γ-phosphates of ATP) by using a 40 or 50 nm Au colloid as a low-load probe. These mutants rotate unidirectionally and the rotation rates obeyed the Michaelis-Menten kinetics. The maximal rotation rates for A(F506E) and B(R350K) were 86 rps and 0.3 rps respectively, and were 0.8 and 0.003 times that of the wild-type (107 rps). The second-order binding rate constant for ATP for A(F506E) and B(R350K) were 5.2 × 104 M−1s−1 and 2.4 × 106 M−1s−1 respectively, and were 0.02 times and equivalent to that of the wild-type (2.3 × 106 M−1s−1). These results strongly suggest that specific interaction of the catalytic site with the adenine ring of ATP is important to accelerate ATP binding, while interaction of arginine-finger with the β- and γ-phosphates of ATP accelerates the ATP hydrolysis.