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 (ENADH), 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 [formula: see text] 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/k-1 = 22.6 microM) was too rapid to be followed by stopped-flow spectrofluorometry at 25 degrees C. The first-order rate constants for reduction (k 2) 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 microM; 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 (k 1) was 2.1 x 10(5) M-1 s-1, and the dissociation rate constant (k-1) was 0.15 s-1; k 2 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.
Porter
D J
DJ
Experimental Therapy Division, Burroughs Wellcome Co., Research Triangle Park, North Carolina 27709.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000378:metabolism*; D043383:Adenosylhomocysteinase; D000426:Alcohol Dehydrogenase / Q000378:metabolism; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D002417:Cattle; D006736:Horses; D006859:Hydrogen / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D008962:Models, Theoretical; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D013379:Substrate Specificity; D014316:Tritium
D001051:Apoenzymes; D009243:NAD; D014316:Tritium; D006859:Hydrogen; D000426:Alcohol Dehydrogenase; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine