TY - JOUR
T1 - F1-ATPase, roles of three catalytic site residues
AU - Au, Sabine Löb
AU - Weber, Joachim
AU - Wilke-Mounts, Susan
AU - Senior, Alan E.
PY - 1997
Y1 - 1997
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0030614445&partnerID=8YFLogxK
U2 - 10.1074/jbc.272.6.3648
DO - 10.1074/jbc.272.6.3648
M3 - Article
C2 - 9013618
AN - SCOPUS:0030614445
SN - 0021-9258
VL - 272
SP - 3648
EP - 3656
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 6
ER -