Open AccessSteady‐state kinetics of horse‐liver alcohol dehydrogenase with a covalently bound coenzyme analogue
Author(s) -
KOVÅŘ Jan,
ŠIMEK Karel,
KUČERA Igor,
MATYSKA Luděk
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb08045.x
Subject(s) - cofactor , nad+ kinase , alcohol dehydrogenase , chemistry , enzyme , nicotinamide adenine dinucleotide , nicotinamide , stereochemistry , kinetics , dehydrogenase , enzyme kinetics , steady state (chemistry) , biochemistry , active site , organic chemistry , physics , quantum mechanics
The steady‐state kinetics of the enzyme modified by affinity labelling with an NAD analogue, nicotinamide‐ N 6 ‐[ N ‐(6‐aminohexyl)carbamoylmethyl]‐adenine dinucleotide, has been investigated using a recycling reaction with p ‐nitrosodimethylaniline and n ‐butanol as substrates and compared to the kinetics of native alcohol dehydrogenase. The modified enzyme obeys a ping‐pong mechanism involving two inactive enzyme forms (enzyme‐NAD and enzyme‐NADH complexes in the ‘open’ conformations, the nicotinamide moieties of the coenzymes being out of the active center). The rate of p ‐nitrosodimethylaniline reduction in the reaction catalyzed by the modified enzyme is comparable to that observed in the presence of the native enzyme. On the other hand, the oxidation of butanol by the modified enzyme is essentially slower under our experimental conditions (pH 8.5). The measurements in the presence of specific alcohol dehydrogenase inhibitors competing with substrates and coenzymes (isobutyramide, pyrazole and AMP) revealed that the relative portion of the inactive ‘open’ form of the enzyme‐NADH complex is negligible, whereas the ‘open’ form of the enzyme‐NAD complex seems to represent a more significant portion (about 30%) under the conditions used.