Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide.
S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.
D043383:Adenosylhomocysteinase; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003028:CME-Carbodiimide / Q000031:analogs & derivatives / Q000378:metabolism*; D002234:Carbodiimides / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008433:Mathematics; D009243:NAD / Q000378:metabolism; D051381:Rats; D012995:Solubility; D014357:Trypsin / Q000378:metabolism; D014867:Water
D000596:Amino Acids; D002234:Carbodiimides; D014867:Water; D009243:NAD; D003028:CME-Carbodiimide; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D014357:Trypsin