Substrate Binding Pattern Research Articles

An alternating site sequence for oxidative phosphorylation suggested by measurement of substrate binding patterns and exchange reaction inhibitions.

Catalysis by beef heart submitochondrial particles of the medium Pi in equilibrium HOH, Pi in equilibrium ATP, and the ATP in equilibrium HOH exchanges is strongly inhibited while the ATPase and intermediate Pi in equilibrium HOH exchange are accelerated when medium ADP is removed by pyruvate kinase action. Arsenate readily blocks completely the Pi in equilibrium ATP and medium Pi in equilibrium HOH exchange reactions, but not the ATP in equilibrium HOH exchange reaction. The residual ATP in equilibrium HOH exchange in presence of arsenate is inhibited by 2,4-dinitrophenol. These results and other data are explained by an alternating site model for oxidative phosphorylation. In this model during net oxidative phosphorylation ATP is formed at one site but is transitorily tightly bound and not released until ADP and Pi bind at a second site and the membrane ATPase complex is energized. Under conditions of net ATP hydrolysis, ATP binding at one site is accompanied by hydrolysis of the transitorily tightly bound ATP as a second site. Attractive features are only one site of input for conformational energization of the membrane ATPase, a single conformational transition that accounts for both the promotion of ADP and Pi binding in a competent mode and the release of tightly bound ATP, and a symmetry of catalytic sites. The Pi in equilibrium ATP exchange is not inhibited by increase in MgADP and MgATP at constant ratios, and the energy-linked ADP in equilibrium ATP exchange is not inhibited by increased concentrations of MgATP and Pi at a constant ratio. Such exchange patterns indicate a random binding and release of ADP and Pi.

Substrate Binding and Reaction Intermediates of Glutamine Synthetase (Escherichia coli W) as Studied by Isotope Exchanges

Abstract Substrate concentration effects on isotopic exchange rates at equilibrium have been measured with Escherichia coli glutamate synthetase (adenylated form) with 32P, 14C, 18O, and 15N. A new test for possible compulsory substrate-binding orders is presented, involving measurement of equilibrium exchange rates while increasing concentration of all substrates. This test shows random substrate-binding patterns for the enzyme. Inhibition of some equilibrium exchange rates while various pairs of substrates were increased in concentration appears to result from competitive rather than compulsory binding order effects. The relative rates of equilibrium exchanges were (glutamate ⇄ glutamine) g (NH3 ⇄ glutamine) g (Pi ⇄ ATP) = (ADP ⇄ ATP). The inequalities show that interconversion of bound substrates is not the only rate-limiting step, and allow deductions about relative association-dissociation rates of various substrates. Glutamine synthetase from E. coli does not catalyze any detectable ADP ⇄ ATP, Pi ⇄ ATP, glutamate ⇄ glutamine, or NH3 ⇄ glutamine exchanges unless all substrates are present. The absence of ADP ⇄ ATP exchange in presence of glutamate or of NH3 ⇄ glutamine exchange in presence of Pi, together with lack of compulsory substrate-binding orders, does not give support to formation of γ-glutamyl phosphate as an enzyme-bound intermediate. No phosphoryl enzyme, glutamyl enzyme, or amido enzyme could be detected by isotopic labeling and isolation procedures. The results suggest further consideration of a concerted reaction mechanism requiring all substrates present in the active site.