TY - JOUR
T1 - Effect of the ε-subunit on nucleotide binding to Escherichia coli F1- ATPase catalytic sites
AU - Weber, Joachim
AU - Dunn, Stanley D.
AU - Senior, Alan E.
PY - 1999/7/2
Y1 - 1999/7/2
N2 - The influence of the ε-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (β-Trp-331). The interaction between ε and F1 was not affected by the mutation. K(d) for binding of ε to βY331W mutant F1 was ~1 nM, and ε inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the ε-depleted and ε-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. K(d1)(MgATP) and K(d1)(MgADP) were an order of magnitude higher in the absence of ε than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the ε-depleted and ε- replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of ε, K(m) equals K(d3). Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (V(max)) MgATP hydrolysis rates.
AB - The influence of the ε-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F1-ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (β-Trp-331). The interaction between ε and F1 was not affected by the mutation. K(d) for binding of ε to βY331W mutant F1 was ~1 nM, and ε inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the ε-depleted and ε-replete forms of the enzyme were those for MgATP and MgADP at the high-affinity catalytic site 1. K(d1)(MgATP) and K(d1)(MgADP) were an order of magnitude higher in the absence of ε than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the ε-depleted and ε- replete enzymes, as were the affinities at all three sites for free ATP and ADP. Comparison of MgATP binding and hydrolysis parameters showed that in the presence as well as the absence of ε, K(m) equals K(d3). Thus, in both cases, all three catalytic binding sites have to be occupied to obtain rapid (V(max)) MgATP hydrolysis rates.
UR - http://www.scopus.com/inward/record.url?scp=0033516336&partnerID=8YFLogxK
U2 - 10.1074/jbc.274.27.19124
DO - 10.1074/jbc.274.27.19124
M3 - Article
C2 - 10383416
AN - SCOPUS:0033516336
SN - 0021-9258
VL - 274
SP - 19124
EP - 19128
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -