Evidence for an essential histidine residue in S-adenosylhomocysteinase from rat liver.
Rat liver S-adenosylhomocysteinase (EC 3.3.1.1) is inactivated by diethyl pyrocarbonate. The inactivation is first order in enzyme and in reagent, and a second-order rate constant of 77 M-1 min-1 is obtained at pH 6.9 and 0 degree C. The rate of inactivation is dependent on pH, and the pH-inactivation rate data show the involvement of a group with a pK of 6.8. The difference spectrum of the inactivated and native enzymes shows a single peak at 242 nm, indicating the modification of histidine residues. No trough at around 280 nm due to O-carbethoxytyrosine is observed. The sulfhydryl content of the enzyme is unchanged by the reaction. The inactivation was reversed by hydroxylamine. Although the reaction with [3H]diethyl pyrocarbonate reveals that a residue(s) other than histidine is (are) also modified, the agreement of the number of histidine residues modified and the number of carbethoxy groups removed by hydroxylamine treatment indicates that the inactivation is solely due to the modification of histidine. Statistical analysis of the residual enzyme activity and the extent of modification shows that, among six modifiable residues per subunit, one which reacts more rapidly with the reagent than the rest is critical for activity. The modified enzyme still retains the capacity to bind adenosine and S-adenosylhomocysteine and to oxidize the 3'-hydroxyl of these compounds as evidenced by the reduction of the enzyme-bound NAD+. Slow but significant exchange of the 4' proton with solvent also occurs with the modified enzyme. Thus, it may be concluded that the histidine residue essential for activity is involved in a catalytic reaction other than the abstraction of 3'-hydroxyl and 4' protons of the substrates.
D000241:Adenosine / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000818:Animals; D002850:Chromatography, Gel; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D006639:Histidine / Q000032:analysis*; D006867:Hydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D051381:Rats; D013050:Spectrometry, Fluorescence
D009243:NAD; D006639:Histidine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine; D004047:Diethyl Pyrocarbonate