Browsing by Author "Muscate, Angelika"
Now showing 1 - 1 of 1
Results Per Page
Sort Options
Item Mechanistic investigations of novel, irreversible inhibitors of S-adenosylhomocysteine hydrolase(1992) Muscate, Angelika; Parry, Ronald J.Isolation of bovine S-adenosylhomocysteine hydrolase by affinity chromatography unexpectedly yielded two forms of the enzyme. Type A contained 4 moles of NAD$\sp+$/mole of enzyme tetramer, while Type B had only half that number of nucleotide cofactors associated with it. The acetylenic analog of adenosine, 9-(5$\sp\prime$,6$\sp\prime$-dideoxy-$\beta$-D-ribo-hex-5$\sp\prime$-ynofuranosyl)-adenine, was synthesized, and its behavior as an inhibitor of Type A enzyme examined. Irreversible inactivation of the enzyme with excess inhibitor was accompanied by the reduction of 2 equivalents of NAD$\sp+$ to NADH, release of the remaining NAD$\sp+$ from the enzyme, and binding of 4 equivalents of inhibitor per enzyme tetramer. Denaturation studies suggested that two equivalents of the inhibitor may form a covalent bond between the enzyme and the oxidized inhibitor. The inactivation behavior exhibited by Type A and Type B enzyme with 2$\sp\prime$-deoxy-2$\sp\prime$,2$\sp\prime$-difluoroadenosine was compared. The Type B enzyme was irreversibly inactivated by excess inhibitor with concurrent reduction of 1 mole of NAD$\sp+$ to NADH and release of 1 mole of NAD$\sp+$. Inactivation of the Type A enzyme was accompanied by the reduction of 1 mole of NAD$\sp+$ to NADH and release of ca. 2 moles of NAD$\sp+$. Upon dialysis, 50% of the original activity of the enzyme was regained. Binding stoichiometries of 1 and 3 moles inhibitor/mole of enzyme tetramer were determined for the Type B and Type A enzyme, respectively. Denaturation studies indicated that 1 equivalent of the oxidized inhibitor may have formed a covalent bond with the Type A enzyme, but not with Type B. The binding behavior of adenosine with both types of the enzyme was also studied. Type B enzyme exhibited a large binding affinity of 4-25 moles of adenosine/mole of enzyme tetramer, while Type A bound only 3 moles of adenosine/mole enzyme tetramer. The binding of adenosine to the Type B enzyme was accompanied by the reduction of 1 equivalent of NAD$\sp+$ to NADH. Denaturation experiments suggested the formation of a covalent bond between the enzyme and one equivalent of adenosine. However, peptide mapping of the reduced enzyme-adenosine complex failed.