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.
Hill
J P
JP
Department of Chemistry and Biochemistry, Massey University, Palmerston North, New Zealand.
Motion
R L
RL
Buckley
P D
PD
Blackwell
L F
LF
D000444:Aldehyde Dehydrogenase / Q000187:drug effects / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology*; D004789:Enzyme Activation; D005453:Fluorescence; D008099:Liver / Q000201:enzymology*; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D012756:Sheep; D020245:p-Chloromercuribenzoic Acid
D000447:Aldehydes; D002729:Chloromercuribenzoates; D009243:NAD; D020245:p-Chloromercuribenzoic Acid; C005556:propionaldehyde; D000444:Aldehyde Dehydrogenase