Ternary complexes of liver alcohol dehydrogenase

Liver alcohol dehydrogenase (LADH; E.C. 1.1.1.1) provides an excellent syst em for probing the role of binding interactions with NAD(+) and alcohols as well as with NADH and the corresponding aldehydes. The enzyme catalyzes th e transfer of hydride ion from an alcohol substrate to the NAD(+) cofactor, yielding the corresponding aldehyde and the reduced cofactor, NADH. The en zyme is also an excellent catalyst for the reverse reaction. X-ray crystall ography has shown that the NAD(+) binds in an extended conformation with a distance of 15 A between the buried reacting carbon of the nicotinamide rin g and the adenine ring near the surface of the horse liver enzyme. A major criticism of X-ray crystallographic studies of enzymes is that they do not provide dynamic information. Such data provide time-averaged and space-aver aged models. Significantly, entries in the protein data bank contain both c oordinates as well as temperature factors. However: enzyme function involve s both dynamics and motion. The motions can be as large as a domain closure such as observed with liver alcohol dehydrogenase or as small as the Vibra tions of certain atoms in the active site where reactions take place. Terna ry complexes produced during the reaction of the enzyme binary entity, E-NA D(+), with retinol (vitamin A alcohol) lead to retinal (vitamin A aldehyde) release and the enzyme binary entity E-NADH. Retinal is further metabolize d via the E-NAD(+)-retinal ternary complex to retinoic acid (vitamin A acid ). To unravel the mechanistic aspects of these transformations, the kinetic s and energetics of interconversion between various ternary complexes are c haracterized. Proton transfers along hydrogen bond bridges and NADH hydride transfers along hydrophobic entities are considered in some detail. Second ary kinetic isotope effects with retinol are not particularly large with th e wild-type form of alcohol dehydrogenase from horse liver. We analyze alco hol dehydrogenase catalysis through a re-examination of the reaction coordi nates. The ground states of the binary and ternary complexes are shown to b e related to the corresponding transition slates through topology and free energy acting along the reaction path. (C) 2001 Elsevier Science Ireland Lt d. All rights reserved.