The effect of p-(chloromercuri)benzoate modification of cytosolic aldehyde dehydrogenase from sheep liver. Evidence for a second aldehyde binding site.

p-Chloromercuribenzoate (PCMB) at stoichiometric levels reacts with a thiol group of the binary NAD+ complex of sheep liver cytoplasmic aldehyde dehydrogenase (E.NAD+) faster than with the corresponding thiol group of either the free enzyme or the binary enzyme. NADH complexes. High concentrations of propionaldehyde have a protective effect against modification of the enzyme with PCMB in steady-state assays. This protection arises from a reduction in the concentration of the E.NAD+ binary complex rather than competition for a common binding site. PCMB has three major effects on aldehyde dehydrogenase. First, rapid reaction with a high-affinity thiol group in the E.NAD+ binary complex causes activation of the steady-state rate. The activation results from an increase in the rate of NADH release from the enzyme. This modification simultaneously protects against dilution-induced dissociation of enzyme tetramers. Second, premodification of the high-affinity thiol group leads to inhibition of the steady-state rate at high propionaldehyde concentrations, because of the increased affinity of the free enzyme for propionaldehyde with the resultant formation of an enzyme-aldehyde dead-end complex. Third, when higher ratios of PCMB to enzyme (> 3:1) are used, one or more other thiol groups are also modified, causing enzyme dissociation and subsequent inactivation. Since modification of the high-affinity thiol by PCMB causes activation, clearly it cannot be the active site acylation center involved in propionaldehyde oxidation. The different amplitudes of the proton burst at high and low propionaldehyde concentrations for the PCMB modified enzyme provide support for a second binding site for propionaldehyde on the enzyme.

Effect of pyrophosphate ions and alkaline pH on the kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase.

Pyrophosphate ions activate the steady-state rate of oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase at alkaline pH values. The steps in the mechanism governing the release of NADH from terminal enzyme. NADH complexes have been shown to be rate-limiting at pH 7.6 [MacGibbon, Buckley & Blackwell (1977) Biochem J. 165, 455-462]. These steps are shown to be also rate-limiting at more alkaline pH values, and it is through an acceleration of these steps that pyrophosphate ions exert their activation effect.

Steady-state and pre-steady-state kinetics of propionaldehyde oxidation by sheep liver cytosolic aldehyde dehydrogenase at pH 5.2. Evidence that the release of NADH remains rate-limiting in the enzyme mechanism at acid pH values.

The kcat value for the oxidation of propionaldehyde by sheep liver cytosolic aldehyde dehydrogenase increased 3-fold, from 0.16 s-1 at pH 7.6 to 0.49 s-1 at pH 5.2, in parallel with the increase in the rate of displacement of NADH from binary enzyme.NADH complexes. A burst in nucleotide fluorescence was observed at all pH values consistent with the rate of isomerization of binary enzyme.NADH complexes constituting the rate-limiting step in the steady state. No substrate activation by propionaldehyde was observed at pH 5.2, but the enzyme exhibited dissociation/association behavior. The inactive dissociated form of the enzyme was favored by low enzyme concentration, low pH, and low ionic strength. Propionaldehyde protected the enzyme against dissociation.

Evidence that the cytoplasmic aldehyde dehydrogenase-catalysed oxidation of aldehydes involves a different active-site group from that which catalyses the hydrolysis of 4-nitrophenyl acetate.

Acylation of the aldehyde dehydrogenase.NADH complex by acetic anhydride leads to the production of acetaldehyde and NAD+. By monitoring changes in nucleotide fluorescence, the rate constant for acylation of the active site of the *enzyme.NADH complex was found to be 11 +/- 3 s-1. The rate of acylation by acetic anhydride at the group that binds aldehydes on the oxidative pathway is clearly rapid enough to maintain significant steady-state concentrations of the required active-site-acylated *enzyme.NADH intermediate despite the rapid hydrolysis of this *enzyme.acyl.NADH intermediate (5-10 s-1) [Blackwell, Motion, MacGibbon, Hardman & Buckley (1987) Biochem. J. 242, 803-808]. Hence reversal of the normal oxidative pathway can occur. However, although acylation of the aldehyde dehydrogenase.NADH complex by 4-nitrophenyl acetate also occurs rapidly with a rate constant of 10.9 +/- 0.6 s-1, even under the most extreme trapping conditions only very small amounts of acetaldehyde are detected [Loomes & Kitson (1986) Biochem. J. 235, 617-619]. Furthermore enzyme-catalysed hydrolysis of 4-nitrophenyl acetate is limited by the rate of deacylation of a group on the enzyme (0.4 s-1), which is an order of magnitude less than deacylation of the group at the active site (5-10 s-1). It is concluded that the enzyme-catalysed 4-nitrophenyl ester hydrolysis involves a group on the enzyme that is different from the active-site group that binds aldehydes on the normal oxidative pathway.

Evidence that the slow conformation change controlling NADH release from the enzyme is rate-limiting during the oxidation of propionaldehyde by aldehyde dehydrogenase.

The displacement of NADH from the aldehyde dehydrogenase X NADH complex by NAD+ was followed at pH 7.0, and the data were fitted by a non-linear least-squares iterative procedure. At pH 7.0 the decay constants for the dissociation of NADH from aldehyde dehydrogenase X NADH complexes (1.62 +/- 0.09 s-1 and 0.25 +/- 0.004 s-1) were similar to the values previously determined by MacGibbon, Buckley & Blackwell [(1977) Biochem. J. 165, 455-462] at pH 7.6, and apparent differences between these values and those reported by Dickinson [(1985) Biochem. J. 225, 159-165] are resolved. Experiments at low concentrations of propionaldehyde show that isomerization of a binary E X NADH complex is part of the normal catalytic mechanism of the enzyme. Evidence is presented that the active-site concentration of aldehyde dehydrogenase is halved when enzyme is pre-diluted to low concentrations before addition of NAD+ and substrate. The consequences of this for the reported values of kcat. are discussed. A general mechanism for the aldehyde dehydrogenase-catalysed oxidation of propionaldehyde which accounts for the published kinetic data, at concentrations of aldehyde which bind only at the active site, is presented.

Activating effect of p-(chloromercuri)benzoate on the cytoplasmic aldehyde dehydrogenase from sheep liver.

In the absence of NAD+, up to 12 SH groups on aldehyde dehydrogenase (ALDH) reacted rapidly with p-(chloromercuri)benzoate (PCMB); a slow reaction with more than twice this number of SH groups then occurred. When PCMB was added to an assay mixture at low (less than 100 microM) concentrations of propionaldehyde, the steady-state rate of production of NADH increased with increasing PCMB concentration up to a maximum activity at a [PCMB]/[ALDH] ratio of 1.9 and then decreased as the [PCMB]/[ALDH] ratio increased further. Under some conditions, activation, or inhibition, showed hysteretic effects as the initial slope after mixing changed to a final linear steady state in a first-order manner, the rate constants for which were proportional to the concentration of free PCMB. Activating levels of PCMB had little effect on the NADH and proton burst amplitudes or rate constants and did not affect the rate of dissociation or association of NADH. However, when a 20-fold excess of PCMB concentration over enzyme concentration was premixed with the enzyme, neither a burst nor a steady-state turnover of substrate was observed. It is concluded that activation arises from the tight binding of PCMB with a single thiol group per subunit which is exposed after the binding of NAD+ to the enzyme, followed by a slow conformational change which causes activation by altering the steady-state mechanism so that NADH dissociation becomes largely rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and properties of sheep-liver aldehyde dehydrogenases.

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min(-1) mg(-1). An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (lambdaexcit = 290 nm, lambdaemisson = 340 nm) is quenched on the binding of NAD+ and NADH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme . NADH complex (Kd = 1.2 +/- 0.2 muM). A Hill plot of the data gave a straight line with a slope of 1.0 +/- 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212 000 +/- 8 000 and 205 000 respectively. Each enzyme consisted of four subunits with molecular weight of 53 000 +/- 2 000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.