Characterization of a transient intermediate formed in the liver alcohol dehydrogenase catalyzed reduction of 3-hydroxy-4-nitrobenzaldehyde
The compounds 3-hydroxy-4-nitrobenzaldehyde and 3-hydroxy-4-nitrobenzyl alcohol are introduced as new chromophoric substrates for probing the catalytic mechanism of horse liver alcohol dehydrogenase (LADH). Ionization of the phenolic hydroxyl group shifts the spectrum of the aldehyde from 360 to 433 nm (pK/sub a/ = 6.0), whereas the spectrum of the alcohol shifts from 350 to 417 nm (pK/sub a/ = 6.9). Rapid-scanning, stopped-flow (RSSF) studies at alkaline pH show that the LADH-catalyzed interconversion of these compounds occurs via the formation of an enzyme-bound intermediate with a blue-shifted spectrum. When reaction is limited to a single turnover of enzyme sites, the formation and decay of the intermediate when aldehyde reacts with enzyme-bound reduced nicotinamide adenine dinucleotide E(NADH) are characterized by two relaxations. Detailed stopped-flow kinetic studies were carried out to investigate the disappearance of aldehyde and NADH, the formation and decay of the intermediate, the displacement of Auramine O by substrate, and /sup 2/H kinetic isotope effects. It was found that (1) NADH oxidation takes place at the rate of the slower relaxation (2) when NADD is substituted for NADH, lambda/sub s/ is subject to a small primary isotope effect; and (3) the events that occur in lambda/sub s/ precede lambda/sub f/. These findings identify the intermediate as a ternary complex containing bound oxidized nicotinamide adenine dinucleotide (NAD/sup +/) and some form of 3-hydroxy-4-nitrobenzyl alcohol. The authors conclude that the LADH substrate site can be divided into two subsites: a highly polar, electropositive subsite in the vicinity of the active-site zinc and, just a few angstroms away, a rather nonpolar region.
- Research Organization:
- Univ. of California, Riverside
- OSTI ID:
- 6024941
- Journal Information:
- Biochemistry; (United States), Vol. 26:11
- Country of Publication:
- United States
- Language:
- English
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HEMIACETAL DEHYDROGENASES
BIOCHEMICAL REACTION KINETICS
ISOTOPE EFFECTS
NITRO COMPOUNDS
ABSORPTION SPECTRA
REDUCTION
REACTION INTERMEDIATES
BENZALDEHYDE
COENZYMES
DEUTERIUM COMPOUNDS
NADH2
ALDEHYDES
CHEMICAL REACTIONS
ENZYMES
HYDROGEN COMPOUNDS
KINETICS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
REACTION KINETICS
SPECTRA
550201* - Biochemistry- Tracer Techniques