S-ADENOSYLHOMOCYSTEINE HYDROLASE - STEREOCHEMISTRY AND KINETICS OF HYDROGEN TRANSFER

The stereochemistry and kinetics for hydrogen transfer to the catalytically essential NAD+ of S-adenosylhomocysteine hydrolase (SAHase) have been determined for selected adenosine analogues. Reduced SAHase (E(NADH)), which was made by reconstituting apoSAHase with NADH, stereospecifically transferred the pro-R hydrogen of NADH to 3'-ketoadenosine, a proposed reaction intermediate. Reduction of SAHase by 5'-deoxyadenosine, alpha-L-lyxofuranosyladenine, or adenosine-5'-carboxylate quenched the intrinsic protein fluorescence and increased the absorbance of the enzyme at 330 nm. The kinetics for reduction were consistent with a two-step mechanism [GRAPHICS] where E.L was an initial complex between ligand (L) and enzyme (E), and E'.L' was a complex between reduced enzyme and oxidized ligand. Initial complex formation between SAHase and alpha-L-lyxofuranosyladenine (k-1/k1 = 22.6 muM) was too rapid to be followed by stopped-flow spectrofluorometry at 25-degrees-C. The first-order rate constants for reduction (k2) and oxidation (k-2) of the enzyme were 7.7 and 0.22 s-1, respectively. The dissociation constant for initial complex formation between 5'-deoxyadenosine and SAHase was 24 muM; k2 and k-2 were 8.4 x 10(-3) and 4.2 x 10(-3) s-1, respectively. The association rate constant for binding of adenosine-5'-carboxylate to SAHase (k1) was 2.1 x 10(5) M-1 s-1, and the dissociation rate constant (k-1) was 0.15 s-1; k2 and k-2 were 0.1 and 0.15 s-1, respectively. The association rate constant for adenine was 5.5 x 10(6) M-1 s-1, and the dissociation rate constant was 6.4 s-1.