Carboxymethylated liver alcohol dehydrogenase: kinetic and thermodynamic characterization of reactions with substrates and inhibitors

Publisher Website
Google Scholar
Abstract
Liver alcohol dehydrogenase (LADH) [horse] carboxymethylated at Cys-46 (CMLADH) forms 2 different ternary complexes with 4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA). The complex with NADH is characterized by a 38-nm red shift of the long-wavelength .pi.,.pi.* transition to 436 nm, while the complex with NAD is characterized by a 60-nm red shift to 458 nm. CMLADH also forms a ternary complex with NAD and the Z isomer of 4-trans-(N,N-dimethylamino)cinnamaldoxime in which the absorption of the oxime (.lambda.max = 354 nm) is red shifted 80 nm to 434 nm. Pyrazole and 4-methylpyrazole are weak competitive inhibitors of ligand binding to the substrate site of native LADH. These inhibitors formed ternary complexes with CMLADH and NADH which are more stable than the corresponding complexes with the native enzyme. The transient reductions of the aldehydes DACA and p-nitrobenzaldehyde (NBZA) were studied under single-turnover conditions. Carboxymethylation decreases the DACA reduction rate 80-fold and renders the process essentially independent of pH over the region 5-9, whereas this process depends on a pKa of 6.0 in the native enzyme. At pH 7.0, the rate constant for NBZA reduction also is decreased at least 80-fold to a value of 7.7 .+-. 0.3 s-1. Since primary kinetic isotope effects are observed when NADH is substituted with (4R)-4-deuterio-NADH (kH/kD = 3.0 for DACA and kH/kD = 2.3 for NBZA), the rate-limiting step for both aldehydes involves hydride transfer. The altered pH dependence is probably due to an increase in the pK value of the zinc-coordinated DACA-alcohol in the ternary complex with NAD by more than 3 U. This perturbation is brought about by the close proximity of the negatively charged carboxymethyl carboxylate.