A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein

Abstract

1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (alpha-epsilon) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the K(d) is of the order of 10(-8)m. 3. The enthalpy of the ATPase-inhibitor complex-formation is positive, the value of K(d) decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase-inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K(2)SO(4), the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase-inhibitor complex concentration. The temperature coefficients of the ;on' and ;off' processes were also measured. 6. A simple kinetic model for the ATPase-inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg(2+), reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.

An ATP dependence of mitochondrial F1‐ATPase inactivation by the natural inhibitor protein agrees with the alternating‐site binding‐change mechanism

“…The ATP dependence of k+l (which is proportional to the 1/C5o value) exhibits a complex pattern ( fig.3) and, at first glance, contradicts the assumption [21][22][23] that some catalytic intermediate of the enzyme is a target for the action of IP. Thus, Panchenko and Vinogradov [24] observed that the rate of IP-induced inactivation of F, was half-saturated at about 5/zM ATP and remained unchanged over the ATP concentration range 0.02-1.0 mM, while F1-ATPase activity was shown to have a Km for ATP of -0.1 mM [25].…”

“…The latter result is in contradiction with the data of Chernyak et al [19] and means that, at least in the case of the active acidic form of IP, Zn 2÷ (and, possibly, Cd 2÷) does not affect the interaction of F1 and IP. The rate of association of IP and F1 was reported to decrease with increasing ionic strength [21]. Since the preincubation medium is of high ionic strength (see section 2), an increase in ionic strength due to a rise in MgATP concentration appears unlikely to be the cause of the decrease in k+~ observed at high ATP concentrations.…”

Proton coupling is preserved in membrane‐bound chloroplast ATPase activated by high concentrations of tentoxin

“…la, compare traces d-e to trace a). It has been known that the rate of FoF~ ATPase deactivation depends on the enzyme turnover (mitochondrial enzyme [26,27], CFoCF ~ [12]). Since the TTX-modified CFoCF ~ hydrolyses ATP 5-6 times slower than the native form, this could explain why the TTX-modified enzyme does not deactivate after ApH+ collapse.…”

The mitochondrial ATP synthase inhibitor protein binds near the C‐terminus of the F1 β‐subunit

“…Its activity, in both synthetic and hydrolytic directions, is inhibited by a naturally occurring, intramitochondrial peptide, IF~ [1], inhibition being complete at 1 mol IF~/mol F1 [2]. Since IF1 is present in a 1 : 1 molar ratio with F1 in bovine heart mitochondria [3], it seems likely that a considerable proportion of F1 exists complexed with IF~ in vivo.…”

“…Like many regulatory peptides (calmodulin [6], troponin C [7], various peptide hormones [8]), IF1 can assume a highly helical configuration with a superficial hydrophobic patch (an 'amphiphilic helix') [9]. Thermodynamic analysis [2] and differential labelling studies [10] have allowed us to identify the binding site for F1 as the hydrophobic patch on this amphiphilic helix. The binding site for IF1 on F1, however, is less well defined.…”