Mechanisms of inactivation of human S-adenosylhomocysteine hydrolase by 5',5',6',6'-tetradehydro-6'-deoxy-6'-halohomoadenosines.
In an effort to design more specific and potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase, we investigated the mechanisms by which 5',5',6', 6'-tetradehydro-6'-deoxy-6'-halohomoadenosines (X = Cl, Br, I) inactivated this enzyme. The 6'-chloro (a) and 6'-bromo (b) acetylenic nucleoside analogues produced partial ( approximately 50%) loss of enzyme activity with a concomitant ( approximately 50%) reduction of E-NAD(+) to E-NADH. In addition, Ade and halide ions were released from the inhibitors in amounts suggestive of a process involving enzyme catalysis. AdoHcy hydrolase, which was inactivated with compound a, was shown to contain 2 mol of the inhibitor covalently bound to Lys318 of two subunits of the homotetramer. These data suggest that the enzyme-mediated water addition at the 5' position of compound a or b produces an alpha-halomethyl ketone intermediate, which is then attacked by a proximal nucleophile (i.e., Lys318) to form the enzyme-inhibitor covalent adduct (lethal event); in a parallel pathway (nonlethal event), addition of water at the 6' position produces an acyl halide, which is released into solution and chemically degrades into Ade, halide ion, and sugar-derived products. In contrast, compound c completely inactivated AdoHcy hydrolase by converting 2 equiv of E-NAD(+) to E-NADH and causing the release of 2 equiv of E-NAD(+) into solution. Four moles of the inhibitor was shown to be tightly bound to the tetrameric enzyme. These data suggest that compound c inactivates AdoHcy hydrolase by a mechanism similar to the acetylenic analogue of Ado described previously by Parry et al. [(1991) Biochemistry 30, 9988-9997].
Yang
X
X
Department of Pharmaceutical Chemistry, The University of Kansas, Lawrence, Kansas 66047, USA.
Yin
D
D
Wnuk
S F
SF
Robins
M J
MJ
Borchardt
R T
RT
D000225:Adenine / Q000378:metabolism; D043383:Adenosylhomocysteinase; D002712:Chlorides / Q000378:metabolism; D003839:Deoxyadenosines / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006219:Halogens / Q000737:chemistry; D006801:Humans; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D009243:NAD / Q000378:metabolism; D010449:Peptide Mapping; D012435:S-Adenosylhomocysteine / Q000378:metabolism*; D016339:Spectrometry, Mass, Fast Atom Bombardment
C417667:5',5',6',6'-tetradehydro-6'-deoxy-6'-bromohomoadenosine; C417666:5',5',6',6'-tetradehydro-6'-deoxy-6'-chlorohomoadenosine; C417668:5',5',6',6'-tetradehydro-6'-deoxy-6'-iodohomoadenosine; D002712:Chlorides; D003839:Deoxyadenosines; D004791:Enzyme Inhibitors; D006219:Halogens; D009243:NAD; D012435:S-Adenosylhomocysteine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000225:Adenine; D008239:Lysine