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)
D000444:Aldehyde Dehydrogenase / Q000378:metabolism*; D000447:Aldehydes; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology*; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD; D012756:Sheep; D020245:p-Chloromercuribenzoic Acid
D000447:Aldehydes; D002729:Chloromercuribenzoates; D009243:NAD; D020245:p-Chloromercuribenzoic Acid; C005556:propionaldehyde; D000444:Aldehyde Dehydrogenase