Rates and equilibriums for the inactivation of muscle aldolase by an active site directed Michael reaction

Abstract

Compound 1,4-(4-nitrophenoxy)-2-keto-n-butylphosphoric acid, was synthesized and shown to undergo elimination ofp-nitrophenol in aqueous solution at pH 7.0 to form 2-keto-3-butenylphosphoric acid ( 2 ) at a rate 50 times greater than hydration to 2-keto-4-hydroxybutylphosphoric acid (4) occurs. A term in the rate expression for 12-4 2 is ascribed to an intramolecular proton abstraction of the 01 proton by the phosphate oxygen. Rabbit muscle aldolase does not catalyze the production of even 1 equiv of p-nitrophenol in the presence of excess 1. The elimination product, 2, however, causes time-dependent inhibition of the enzyme. The pH and concentration dependence of this reaction was studied, as well as protection from inhibition by 2 using dihydroxyacetone phosphate. Formation of the mixed disulfide of 6-mercaptopropionate with an active-site thiol group also affords protection from inhibition by 2. The rate of hydrolysis of the enzyme-inhibitor complex was measured by two methods, and this demonstrated that the equilibrium constant for binding of 2 was IO9 M-' at pH 7. Compounds 1 and 4 were shown to bind to aldolase but not undergo reaction. A mechanism for inactivation is proposed in which 2 forms an iminium ion with the lysine function, followed by a Michael-type addition of a thiol to afford the inactive complex. IntroductionWe have previous:;. shown' that 2-keto-3-but en ylphospha te (2) inhibits rabbit muscle aldolase in an active site directed, time-dependent manner. A logical explanation of this behavior2 is that 2 condenses with the €-amino group of lysine present at the active site,3 followed by attack of an active-site nucleophile, as shown in Scheme I. The formation of the iminium ion would be analogous to the condensation of the natural substrate dihydroxyacetone phosphate (DHAP) with the enzyme. Aldolase does not catalyze the steady-state production of p-nitrophenol from 1, even though a reasonable mechanism may be written using precedentedIn this paper we describe the details of the syntheses and reactivity of 1,2, and 4 and their interactions with the enzyme. In particular, we wished to determine if a single turnover of 1 to 2 was catalyzed by aldolase and also to determine if the inhibition by 2 was reversible. It was also desirable to see if an active site directed thiol reagent would prevent inhibition in order to determine if a cysteine could be implicated as the reacting nucleophile.A study of both the forward and reverse rates of reaction might provide some generally useful information about the free energy of interaction covalent inhibitors with enzymes. Many of the suicide type of inhibitors or those that react further after a Michael-type reaction would not be amenable to study in

Latent inhibitors. Part 2. Allylic inhibitors of alcohol dehydrogenase

“…A series of inhibitors covering a range of leaving group abilities was synthesised and tested against HLADH at pH 9, the optimum pH for alcohol oxidation. The structures and inhibitory properties of the compounds 8)-( 10) and ( 13 Inhibition of HLADH by the Tkioethers (11) and (12).-…”

“…The requirement for the catalytic intervention of the enzyme was demonstrated initially by the fact that NAD+ is essential for activation (Figure 1). Further, it was shown that oxidation of analogous to the alcohol, but at a rate too fast for 1,l-dideuterio- ( 11) was 1.4 times slower than that of the measurement by conventional, spectrophotometric unlabelled compound and also that inhibition is cor-techniques. respondingly slower in the former case (Figure 2).…”

“…I t is interesting to estimate the fraction of turnovers Evidence for the kinetic order of the reaction is pre-by the enzyme that gives rise to inactivation and the 3. Inhibition could be prevented by the addition of ethanol which competes with (11) for the active site of HLADH. A solvolysis product of (11) is not involved because preincubation of (11) with NAD+ causes no initial inhibition of the enzyme.…”

“…Inhibition could be prevented by the addition of ethanol which competes with (11) for the active site of HLADH. A solvolysis product of (11) is not involved because preincubation of (11) with NAD+ causes no initial inhibition of the enzyme. Together, these results indicate that 3-ethylthioprop-2-en-1-01 is an effective latent inhibitor of HLADH and imply that the corresponding aldehyde (12) should also be an inhibitor.…”

“…A limitation to the use of the ethylthioallyl derivatives became apparent when it was observed that, on prolonged standing for several days after inhibition by either the thioether (11) or (12), HLADH gradually…”