Mechanism of bovine liver S-adenosylhomocysteine hydrolase. Steady-state and pre-steady-state kinetic analysis.
The kinetic mechanism of S-adenosylhomocysteine hydrolase was investigated by stopped-flow spectrofluorometry at pH 7.0 and 25 degrees C. Pre-steady-state kinetic steps were identified with chemical steps proposed for the mechanism of this enzyme (Palmer, J.L., and Abeles, R.H. (1979) J. Biol. Chem. 254, 1217-1226). The steady-state kinetic constants for the hydrolysis or synthesis of S-adenosylhomocysteine were in good agreement with those values calculated from the pre-steady-state rate constants. The equilibrium constant for dehydration of 3'-ketoadenosine to 3'-keto-4',5'-dehydroadenosine on the enzyme was 3. The analogous equilibrium constant for addition of L-homocysteine to S-3'-keto-4',5'-dehydroadenosylhomocysteine on the enzyme was 0.3. The elimination of H2O from adenosine in solution had an equilibrium constant of 1.4 (aH2O = 1). Thus, the equilibrium constants for these elimination reactions on the enzyme were probably not perturbed significantly from those in solution. The equilibrium constant for the reduction of enzyme-bound NAD+ by adenosine was 8, and the analogous constant for the reduction of the enzyme by S-adenosylhomocysteine was 4. The equilibrium constant for the reduction of NAD+ by a secondary alcohol in solution was 5 x 10(-5) at pH 7.0. Consequently, the reduction of enzyme-bound NAD+ by adenosine was 10(5)-fold more favorable than the reduction of free NAD+. The magnitude of the first-order rate constants for the interconversion of enzyme-bound intermediates varied over a relatively small range (3-80 s-1). Similarly, the magnitude of the equilibrium constants among enzyme-bound intermediates varied over a narrow range (0.3-10). These results were consistent with the overall reversibility of the reaction.
Porter
D J
DJ
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Boyd
F L
FL
D000241:Adenosine / Q000031:analogs & derivatives / Q000138:chemical synthesis / Q000378:metabolism; D043383:Adenosylhomocysteinase; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002852:Chromatography, Ion Exchange; D006867:Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008433:Mathematics; D008962:Models, Theoretical; D013050:Spectrometry, Fluorescence
D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine