Horse Liver Alcohol Dehydrogenase
Year: 1973
Type: article
Abstract: Picolinimidylation of one amino group per active site of horse liver alcohol dehydrogenase increases the turnover numbers of the enzyme approximately 10-fold and other kinetic constants up to 140-fold at pH 8. The picolinimidylated enzyme shifts the absorption maximum of NADH to lower wave lengths, indicating that the amino group that can be modified is probably not involved in the spectral shift observed when NADH binds to native enzyme. Product and dead-end inhibition studies at pH 8 indicate that the mechanism of the modified enzyme conforms to an Ordered Bi Bi and not to a rapid equilibrium random mechanism. Stopped flow studies show that NAD+ binds 10 times more slowly to the modified enzyme than to the native enzyme and that NADH binds at about the same rate to both enzymes. Dissociation of NADH (5.6 s-1) is rate-limiting for the reaction of NAD+ and ethanol catalyzed by the native enzyme but was too fast to measure (g200 s-1) with the modified enzyme. The transfer of hydrogen from ethanol to NAD+ now controls the turnover (32 s-1) with the modified enzyme as shown by a deuterium isotope effect of 4.8 in the steady state reaction. The maximum velocity of the reaction of NAD+ and ethanol is dependent upon a group with a pK of 7.2; this group must be unprotonated for maximal activity and could be an imidazole. For the reaction of NADH and acetaldehyde, no isotope effect was observed and the turnover (550 s-1) may be limited by isomerization and dissociation of the enzyme-NAD+ complex.
Source: Journal of Biological Chemistry
Authors: Bryce V. Plapp, Robert L. Brooks, Joseph D. Shore
Institutions: University of Iowa, Henry Ford Hospital
Language: English
Topic: Chemical Reaction Mechanisms
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $3,430
OpenAlex ID: W1584109119