Inactivation of rat liver S-adenosylhomocysteinase by iodoacetamide.
S-Adenosylhomocysteine (EC 3.3.1.1) from rat liver is inactivated by iodoacetamide following pseudo-first-order reaction kinetics. The apparent first-order rate constant for inactivation is proportional to the concentration of the modifier, and a value of 7.55 M-1 min-1 is obtained for the second-order rate constant at pH 9.06 and 25 degrees C. Amino acid analysis of the modifier enzyme shows the formation of S-(carboxymethyl)cysteine. No peaks corresponding to N epsilon-(carboxymethyl)- and N epsilon,N epsilon-bis(carboxymethyl)lysines, N-(carboxymethyl)histidines, S-(carboxymethyl)homocysteine, homoserine, and homoserine lactone are detected. Glycolic acid is also not found in the acid hydrolysate of the modified enzyme, indicating the absence of modification at carboxyl residues. These results and the finding that the number of residues modified as determined by the incorporation of iodo[1-14C]acetamide is equal to the number of cysteine residues lost by modification establish the site of modification as cysteine residues. Kinetics of inactivation and incorporation of the label from iodo[1-14C]acetamide show that two among three modifiable residues per enzyme subunit are essential for activity and the modification of either results in complete inactivation. The inactivation by iodoacetamide does not involve alteration in the molecular size of enzyme nor release of the bound NAD+. The modified enzyme still retains the capacity to bind adenosine and to oxidize it as evidenced by the reduction of enzyme-bound NAD+ but does not catalyze the exchange of the 4' proton with solvent. Thus, it is suggested that the inability of the modified enzyme to catalyze the overall reaction is due to the failure to abstract the 4' proton in the catalytic cycle.
D000225:Adenine / Q000494:pharmacology; D000241:Adenosine / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000596:Amino Acids; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D006867:Hydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D007460:Iodoacetamide / Q000494:pharmacology*; D007461:Iodoacetates / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D051381:Rats; D013438:Sulfhydryl Compounds
D000596:Amino Acids; D007461:Iodoacetates; D013438:Sulfhydryl Compounds; D009243:NAD; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000225:Adenine; D000241:Adenosine; D007460:Iodoacetamide