Specific tryptophan substitution in catalytic sites of Escherichia coli F1-ATPase allows differentiation between bound substrate ATP and product ADP in steady-state catalysis

Joachim Weber, Cheryl Bowman, Alan E. Senior

Research output: Contribution to journalArticle

59 Scopus citations

Abstract

Tryptophan was specifically inserted as the residue immediately preceding the P-loop sequence in F1-ATPase catalytic sites. The mutant enzyme (βF148W) showed normal enzymatic characteristics. The fluorescence responses of β-tryptophan 148 enabled us to differentiate between nucleoside di- and triphosphate bound in catalytic sites; MgADP quenched at 350 nm, whereas MgAMPPNP and MgADP·BeF(x) 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 F1-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.

Original languageEnglish
Pages (from-to)18711-18718
Number of pages8
JournalJournal of Biological Chemistry
Volume271
Issue number31
DOIs
StatePublished - 1996

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