THE EFFECT OF P-(CHLOROMERCURI)BENZOATE MODIFICATION OF CYTOSOLIC ALDEHYDE DEHYDROGENASE FROM SHEEP LIVER - EVIDENCE FOR A 2ND-ALDEHYDE BINDING-SITE

p-Chloromercuribenzoate (PCMB) at stoichiometric levels reacts with a thiol group of the binary NAD+ complex of sheep liver cytoplasmic alde hyde 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 agai nst 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. PC MB has three major effects on aldehyde dehydrogenase. First, rapid rea ction with a high-affinity thiol group in the E . NAD+ binary complex causes activation of the steady-state rate. The activation results fro m an increase in the rate of NADH release from the enzyme. This modifi cation simultaneously protects against dilution-induced dissociation o f enzyme tetramers. Second, premodification of the high-affinity thiol group leads to inhibition of the steady-state rate at high propionald ehyde concentrations, because of the increased affinity of the free en zyme for propionaldehyde with the resultant formation of an enzyme-ald ehyde dead-end complex. Third, when higher ratios of PCMB to enzyme (> 3:1) are used, one or more other thiol groups are also modified, causi ng enzyme dissociation and subsequent inactivation. Since modification of the high-affinity thiol by PCMB causes activation, clearly it cann ot be the active site acylation center involved in propionaldehyde oxi dation. The different amplitudes of the proton burst at high and low p ropionaldehyde concentrations for the PCMB modified enzyme provide sup port for a second binding site for propionaldehyde on the enzyme. (C) 1994 Academic Press, Inc.