F1-ATPase, roles of three catalytic site residues

Sabine Löb Au, Joachim Weber, Susan Wilke-Mounts, Alan E. Senior

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

Three critical residues, β-Lys-155, β-Asp-242, and β-Glu-181, situated close to the γ-phosphate of MgATP in F1-ATPase catalytic sites, were investigated. The mutations βK155Q, βD242N, and βE181Q were each combined with the βY331W mutation; the fluorescence signal of β-Trp-331 was used to determine MgATP, MgADP, ATP, and ADP binding parameters for the three catalytic sites of the enzyme. The quantitative contribution of side chains to binding energy at all three catalytic sites was calculated. The following conclusions were made. The major functional interaction of β-Lys-155 is with the γ-phosphate of MgATP and is of primary importance at site 1 (the site of highest affinity) and site 2. Release of MgATP during oxidative phosphorylation requires conformational re-positioning of this residue. The major functional interaction of β-Asp-242 is with the magnesium of the magnesium nucleotide at site 1; it has little or no influence at site 2 or 3. In steady-state turnover, the MgATP hydrolysis reaction occurs at site 1. β- Glu-181 contributes little to nucleotide binding; its major catalytic effect derives apparently from a role in reaction chemistry per se. This work also emphasizes that nucleotide binding cooperativity shown by the three catalytic sites toward MgATP and MgADP is absolutely dependent on the presence of magnesium.

Original languageEnglish
Pages (from-to)3648-3656
Number of pages9
JournalJournal of Biological Chemistry
Volume272
Issue number6
DOIs
StatePublished - 1997

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