Key Chemical Factors of Arginine Finger Catalysis of F₁-ATPase Clarified by an Unnatural Amino Acid Mutation

Abstract

A catalytically important arginine, called Arg finger, is employed in many enzymes to regulate their functions through enzymatic hydrolysis of nucleotide triphosphates. F1-ATPase (F1), a rotary motor protein, possesses Arg fingers which catalyze hydrolysis of adenosine triphosphate (ATP) for efficient chemomechanical energy conversion. In this study, we examined the Arg finger catalysis by single-molecule measurements for a mutant of F1 in which the Arg finger is substituted with an unnatural amino acid of a lysine analogue, 2,7-diaminoheptanoic acid (Lyk). The use of Lyk, of which the side chain is elongated by one CH2 unit so that its chain length to the terminal nitrogen of amine is set to be equal to that of arginine, allowed us to resolve key chemical factors in the Arg finger catalysis, i.e., chain length matching and chemical properties of the terminal groups. Rate measurements by single-molecule observations showed that the chain length matching of the side-chain length is not a sole requirement for the Arg finger to catalyze the ATP hydrolysis reaction step, indicating the crucial importance of chemical properties of the terminal guanidinium group in the Arg finger catalysis. On the other hand, the Lyk mutation prevented severe formation of an ADP inhibited state observed for a lysine mutant and even improved the avoidance of inhibition compared with the wild-type F1. The present study demonstrated that incorporation of unnatural amino acids can widely extend with its high "chemical" resolution biochemical approaches for elucidation of the molecular mechanism of protein functions and furnishing novel characteristics.

In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits

“…In the course of these changes, a conserved α subunit arginine inserts into the nucleotide binding pocket of the β subunit (7). Substitution of the arginine residue inhibits ATP production, highlighting the essential role of the α subunit in catalysis (8,9).…”

Construction of Asymmetrical Hexameric Biomimetic Motors with Continuous Single-Directional Motion by Sequential Coordination

“…In the ASCE (additional strand catalytic E) ATPase superfamily, one well‐conserved ATP domain contains one arginine finger motif along with the Walker A and Walker B motifs . Located in proximity to the γ‐phosphate of the bound ATP in the adjacent ATPase subunit, arginine finger has been reported to be involved in the formation of the ATP activity pocket . Elucidation of the principle of how arginine finger mediates the sequential action of ATPase has shed light on the realization of controlling motor motion direction.…”

Chemo-mechanical Coupling in the Transport Cycle of a Type II ABC Transporter

“…Oldham and Chen 61 have recently pointed out the role of the serine residues in coupling conformational change of a type I maltose ABC transporter to its catalytic activity, and that its role resembles that of the “arginine finger” of the biomolecular motor F1-ATPase, which controls the catalytic activity of the protein. 62 However, to our knowledge, the mechanism, by which the wasteful hydrolysis of ATP in the Occ state is avoided, has not yet been addressed for any ABC transporters. Here, by combined use of structural modeling and MD simulation, the mechanism of chemo-mechanical coupling in the type II ABC transporter can be explained in atomistic detail.…”