Purification of S-adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum: reversible inactivation by cAMP and 2'-deoxyadenosine.
S-Adenosyl-L-homocysteine hydrolase from Dictyostelium discoideum has been purified to homogeneity. It is composed of four subunits, each with a molecular mass of 47,000. In the hydrolysis direction, the enzyme has a pH optimum of 7.5, a Km for S-adenosyl-L-homocysteine (SAH) of 6 microM, and a Vmax of 0.22 mumol min-1 mg-1. In the synthesis direction, the pH optimum is 8.0, the Km for adenosine is 0.4 microM, and the Vmax is 0.30 mumol min-1 mg-1. Although the enzyme binds beta-nicotinamide adenine dinucleotide, as well as adenosine 3',5'-cyclic monophosphate and 2'-deoxyadenosine, these ligands have no effect on enzymatic activity when added to the assay mixture. However, preincubation of SAH hydrolase with NAD+ results in a 25% activation of the enzyme. In addition, this ligand has a striking effect on subunit-subunit interactions, as shown by stabilization of quaternary structure during polyacrylamide gel electrophoresis. Preincubation with cAMP or 2'-deoxyadenosine inactivates the enzyme. Although in both cases the activity is restored upon further incubation with NAD+, we show that inactivation by these two ligands proceeds by different mechanisms. NAD+-reversible inactivation by cAMP and 2'-deoxyadenosine was also observed with the SAH hydrolase from rabbit erythrocytes. Thus, these previously unreported properties of SAH hydrolase also occur with mammalian enzymes and are not restricted to D. discoideum.
D043383:Adenosylhomocysteinase; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D000242:Cyclic AMP / Q000494:pharmacology*; D003839:Deoxyadenosines / Q000494:pharmacology*; D004023:Dictyostelium / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006868:Hydrolysis; D009243:NAD / Q000494:pharmacology; D010446:Peptide Fragments
D003839:Deoxyadenosines; D004793:Enzyme Reactivators; D010446:Peptide Fragments; D009243:NAD; D000242:Cyclic AMP; D006867:Hydrolases; D043383:Adenosylhomocysteinase