L L Kosley | PubMed Health

PubMed1998

N-tosyl-L-phenylalanine chloromethyl ketone, a serine protease inhibitor, identifies glutamate 398 at the coenzyme-binding site of human aldehyde dehydrogenase. Evidence for a second "naked anion" at the active site.

Human aldehyde dehydrogenase isozymes were inactivated by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin. The inactivation was a first-order process that followed saturation kinetics. NAD and chloral when used together protected against inactivation. In steady-state kinetics, TPCK produced only slope effects versus varied NAD, both slope and intercept effects versus varied glycolaldehyde were produced, indicating that TPCK reacted with the same enzyme form with which NAD reacted. Ki values from steady-state kinetics and saturation kinetics were comparable. Use of [3H]-labeled TPCK showed that inactivation was associated with the incorporation of two molecules of TPCK per molecule of enzyme. The label incorporation occurred into a single tryptic peptide and also into a single chymotryptic peptide of the E1 isozyme. Purification of labeled peptides, followed by sequencing, demonstrated that E398 of aldehyde dehydrogenase was labeled. Reaction of a haloketone, TPCK, with a carboxyl group of E398 indicates that E398 occurs as a "naked anion" within the molecule. This paper constitutes identification of the second (after E268) "naked anion" at the active site of aldehyde dehydrogenase.

Aldehyde Dehydrogenase↗

PubMed1989

Human aldehyde dehydrogenase: coenzyme binding studies.

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Dehydrogenase↗