Reduced S-adenosylhomocysteine hydrolase. Kinetics and thermodynamics for binding of 3'-ketoadenosine, adenosine, and adenine.
S-Adenosylhomocysteine hydrolase (SAHase) was resolved into apoenzyme and NAD+ by acidic ammonium sulfate treatment. The apoenzyme was catalytically inactive, but could be reconstituted to active enzyme with NAD+. Reduced SAHase (ENADH) that was prepared by reconstitution of the apoenzyme with NADH was catalytically inactive. ENADH was oxidized by 3'-ketoadenosine to active SAHase. The recovery of activity paralleled the oxidation of enzyme-bound NADH. The association rate constant for ENADH and 3'-ketoadenosine was 6.1 x 10(2) M-1 s-1, and the dissociation rate constant was calculated to be 4 x 10(-7) s-1. This association rate constant was considerably smaller than the association rate constant for adenosine and SAHase (greater than 10(7) M-1 s-1). However, the observed pseudo first-order rate constant for reaction of 3'-ketoadenosine with ENADH (0.6 s-1 with 1 mM 3'-ketoadenosine) approached kcat for the hydrolytic reaction (1.2 s-1). Thus, bound 3'-ketoadenosine probably reacted sufficiently rapidly with ENADH to be considered a kinetically competent intermediate. The dissociation constants of SAHase for adenosine and 4',5'-dehydroadenosine, substrates for the enzyme, were 9 and 14 microM, respectively. In contrast, the dissociation constants of ENADH for 3'-ketoadenosine and 4',5'-dehydro-3'-ketoadenosine, intermediates of the catalytic reaction, were significantly lower with values of 600 and 300 pM, respectively. The equilibrium constant for reduction of enzyme-bound NAD+ in the absence of an adenosine analogue, as estimated from cyanide binding studies, was 10-fold more favorable than that for free NAD+. ENADH was highly fluorescent (emission maximum 428 nm, excitation 340 nm) with a quantum yield that was six times that of free NADH. Since SAHase reduced by adenosine was not highly fluorescent, enzyme-bound intermediates quenched the fluorescence of enzyme-bound NADH. Adenosine and adenine quenched the fluorescence of ENADH. Cyanide formed a complex with SAHase that was analogous to ENADH. Adenine stabilized this complex sufficiently that addition of 65 microM adenine and 25 mM cyanide to SAHase caused total complex formation with loss of over 95% of the catalytic activity.
Porter
D J
DJ
Experimental Therapy Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709.
Boyd
F L
FL
D000225:Adenine / Q000378:metabolism*; D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D043383:Adenosylhomocysteinase; D000818:Animals; D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D006867:Hydrolases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D008962:Models, Theoretical; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics
D001051:Apoenzymes; D009243:NAD; C072837:3'-ketoadenosine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000225:Adenine; D000241:Adenosine