Effect of the ε-Subunit on Nucleotide Binding toEscherichia coli F1-ATPase Catalytic Sites

Abstract

The influence of the ⑀-subunit on the nucleotide binding affinities of the three catalytic sites of Escherichia coli F 1 -ATPase was investigated, using a genetically engineered Trp probe in the adenine-binding subdomain (␤-Trp-331). The interaction between ⑀ and F 1 was not affected by the mutation. K d for binding of ⑀ to ␤Y331W mutant F 1 was ϳ1 nM, and ⑀ inhibited ATPase activity by 90%. The only nucleotide binding affinities that showed significant differences in the ⑀-depleted and ⑀-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 ⑀ than in its presence. In contrast, the binding affinities for MgATP and MgADP at sites 2 and 3 were similar in the ⑀-depleted and ⑀-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 ⑀, 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.

Bi-site Catalysis in F1-ATPase: Does It Exist?

“…As our F 1 preparations themselves are largely ⑀-replete (Ն90%; see Ref. 16), and as the concentrations of F 1 used in all experiments (Ն80 nM) were 2 orders of magnitude higher than K d for ⑀ binding (about 1 nM; see Ref. 16), the resulting enzyme was Ͼ99% ⑀-replete.…”

“…By comparing the MgATP concentration dependence of nucleotide binding and hydrolytic activity in ␤Y331W F 1 , we were able to determine that steady-state hydrolytic activity was at least largely due to enzyme molecules with all three catalytic sites occupied (1,12). In a number of analyses using this method, the contribution of bi-site catalysis was estimated to be between 0 and 5% of V max (12,15,16).…”

“…By comparing the MgATP concentration dependence of nucleotide binding and hydrolytic activity in ␤Y331W F 1 , we were able to determine that steady-state hydrolytic activity was at least largely due to enzyme molecules with all three catalytic sites occupied (1,12). In a number of analyses using this method, the contribution of bi-site catalysis was estimated to be between 0 and 5% of V max (12,15,16).If bi-site catalysis were a true working mode of F 1 and could serve as an experimental model for multisite catalysis, it would clearly facilitate analysis of the enzymatic mechanism, particularly rotation. Thus, we decided to re-address the question of the extent of bi-site catalysis, putting emphasis on analyses in the substrate concentration range where enzyme species with two sites filled are most prevalent.…”

“…Thus, we decided to re-address the question of the extent of bi-site catalysis, putting emphasis on analyses in the substrate concentration range where enzyme species with two sites filled are most prevalent. A prerequisite for this analysis is the exact knowledge of substrate binding affinities of the three catalytic sites, to allow calculation of the distribution of all enzyme species at any given substrate concentration.In previous work (12,15,16), MgATP had been used as substrate. It has the technical disadvantage of having an overall high affinity for the catalytic sites of F 1 , and so K d1 and K d2 are in a range where the true concentration of free substrate, knowledge of which is necessary to calculate the binding constants, is difficult to determine precisely.…”

“…In previous work (12,15,16), MgATP had been used as substrate. It has the technical disadvantage of having an overall high affinity for the catalytic sites of F 1 , and so K d1 and K d2 are in a range where the true concentration of free substrate, knowledge of which is necessary to calculate the binding constants, is difficult to determine precisely.…”