Specific tryptophan substitution in catalytic sites of Escherichia coli F-1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis

Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F-1-ATPase catalytic sites. The mutant enzyme (beta F148W) showed normal enzymatic characteristics. The fluorescence responses of beta-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP . BeFx complex enhanced the fluorescence at 325 nm. With MgATP, both effects were seen simultaneously. This allowed analysis of bound catalytic site nucleotides directly under steady-state MgATP hydrolysis conditions. At mM concentration of MgATP (V-max conditions) one of the three catalytic sites was filled with substrate MgATP and the other two sites were filled with product MgADP. A model for F-1-ATPase steady-state turnover is presented that encompasses these findings. Given the structural similarity of the P-loop in nucleotide-binding proteins, this approach may prove widely useful.