A kinetic study of the interaction between mitochondrial F1 adenosine triphosphatase and adenylyl imidodiphosphate and guanylyl imidodiphosphate

Abstract

1. The presence of 5'-adenylyl imidodiphosphate, a non-hydrolysable analogue of ATP, in the solution used to assay the soluble bovine heart mitochondrial F1-ATPase produced slow competitive inhibition. If the enzyme was preincubated with the inhibitor before the substrate, MgATP, was added, a partial re-activation was obtained. 2. The slow inhibitory process showed first-order rate kinetics, and therefore it seems likely that a conformational change of the enzyme occurs following a faster binding process. A reaction scheme is suggested. At pH 7.8 the rate constant for the inhibition reaction was calculated to be 6.7 X 10(-2)s-1 and that for the re-activation 3.8 X 10(-3)s-1, with Keq. 17.6, indicating that the inhibited enzyme-inhibitor complex will be favoured over the non-inhibited enzyme-inhibitor complex. 3. The presence of 5'-guanylyl imidodiphosphate in the solution used to assay F1-ATPase produced rapid competitive inhibition, which was then slowly reversed until a steady state was reached. This might be explained by a rapid but reversible shift of the inhibition pathway induced by this non-hydrolysable analogue of ATP. A complex rate constant for the displacement of the inhibitor by the substrate of 7.6 X 10(-3)s-1 was calculated. 4. The results are discussed in the light of other recent observations about binding of 5'-adenylyl imidodiphosphate to F1-ATPase and with reference to the binding-site-change mechanism of hydrolysis of ATP by F1-ATPase.

4-Substituted Resorcinols (Sulfite Alternatives) as Slow-Binding Inhibitors of Tyrosinase Catecholase Activity

“…The oxy form is an obligatory intermediate in the catalytic turnover, and thus, the presence of the substrate (and therefore of the catalytic activity) is necessary for the slow binding of inhibitor to the enzyme to be observed. For this reason 4-substituted resorcinols cannot be considered as classical competitive inhibitors and are also different from other previously described slow-binding inhibitors (Belda et al, 1983), since 4-substituted resorcinols require an enzymatic turnover for their inhibitory effect to be exhibited.…”

Dimethyl Sulfide, a Volatile Flavor Constituent, Is a Slow-Binding Inhibitor of Tyrosinase

“…Thus, DMS competes with the second molecule of L-DOPA to bind to the oxy form of the enzyme. For this reason DMS cannot be considered as a classical competitive inhibitor (36) and is also different from other previously described slow-binding inhibitors (37), since DMS requires an enzymatic turnover to exhibit its inhibitory effect.…”

Slow Binding Inhibition of S-Adenosylmethionine Synthetase by Imidophosphate Analogues of an Intermediate and Product

“…Thus the slow binding behavior of PNPNP probably arises from steps after the initial binding event, steps which depend on the presence of AdoMet. The inhibition of phosphoryl transferases by imidophosphate analogues have been long exploited due to the resistance to cleavage of the P−N bonds ( − ). Structurally PNP, and presumably other imidopolyphosphates, are essentially isosteric with their oxy analogues ( , ) .…”