THE REACTION RATE OF ENZYMES ON LIVER

The Reaction Rate of Enzymes on Liver



The Reaction Rate of Enzymes on Liver

Introduction and Background of the Research

A considerable body of evidence, as reviewed by Sund and Theorell and supported by more recent studies, shows that in catalysis by liver alcohol dehydrogenase in the presence of excess reactants, dissociation of oxidized or reduced coenzyme is rate-limiting. Sund and Theorell concluded that all four binary complexes between enzyme and nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide, alcohol, or acetaldehyde may form (I). On this basis, the mechanism may be described as kinetically ordered but not strictly compulsory. There is not, however, clear agreement on whether all binary complexes exist. Dalziel, from studies with coenzyme preparations free from inhibitors, concluded that there is no definite evidence for kinetically significant binary complexes with alcohol and acetaldehyde (Baraona & Abittan et al. 2001: 502). Furthermore, McKinley-McKee, on the basis of fluorometric evaluation of possible combinations with acetaldehyde and ethanol, concluded that the binding sites for substrates do not exist unless coenzyme forms are bound.

In contrast to the liver enzyme, most kinetic and related studies with yeast alcohol dehydrogenase have been regarded as showing a rate-limiting interconversion of ternary complexes with random dissociation of substrates and coenzymes. Product inhibition studies by Wratten and Cleland, however, are not in accord with such a mechanism. Recent theoretical and experimental developments have shown that measurement of enzymic reaction rates at equilibrium may be of considerable value in assessing the possible existence of compulsory orders of substrate binding and of ternary complex interconversion as a rate-limiting step. Results with lactate dehydrogenases are reported in the preceding paper (Baraona, 2000: 405). Application of this approach to the liver and yeast alcohol dehydrogenase reactions thus seemed particularly pertinent. The results, as reported herein, show that the reaction with both dehydrogenases does not proceed with a strictly compulsory order of substrate binding, and thus that all four binary complexes exist. Also, for both dehydrogenases, coenzyme dissociation from ternary complexes is slow compared to alcohol or acetaldehyde dissociation, and ternary complex interconversion is not rate-limiting. These studies also reveal other facets of the mechanism not ascertained by other approaches.

Experimental Procedure

Special Reagents-NAD and disodium NADH were obtained from Pabst Laboratories, Milwaukee, and Sigma Chemical Company, St. Louis; DEAE-cellulose from Sigma Chemical Company; 2,4-dinitrophenylhydrazine from Eastman Organic Chemicals, Rochester, New York; and Triton X-100, a nonionic detergent, from Rohm and Haas, Philadelphia. Nicotinamide- (carboxy&1% (6.5 mc per mmole) and ethanol-1-14C (5 mc per mmole) were from California Corporation for Biochemical Research, Los Angeles, and acetaldehyde-1,2-r4C (1 mc per mmole) from Volk Radiochemical Company, Chicago. A purified crystalline suspension (in ammonium sulfate) of liver alcohol dehydrogenase was obtained from Worthington Biochemical Corporation, Freehold, New Jersey, and of yeast alcohol dehydrogenase from Boehringer und Soehne, Mannheim, Germany. Lyophilized yeast alcohol dehydrogenase was obtained from Worthington Biochemical Corporation (Bailey & Cunningham, 1998: 1318). Shortly prior to use, enzyme crystals were removed by centrifugation and dissolved in 35 mM diethylbarbiturate ...