Function and reactivity of sulfhydryl groups of rat liver glycine methyltransferase.
Rat liver glycine methyltransferase is completely inactivated by 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Treatment of the inactivated enzyme with KCN results in a reactivated enzyme having values of Vmax and S0.5 for S-adenosyl-L-methionine comparable to those of the native enzyme and about a 4-fold greater Km value for glycine. Kinetics of inactivation and reactivation show that one cysteine residue is involved in this process. Reaction of the methyltransferase with iodoacetate leads to partial inactivation of the enzyme; about 22% of the initial activity is retained in the modified enzyme. The relationship between the loss of enzyme activity and the number of iodoacetate molecules incorporated and the sequence analysis of peptides containing the modified residues indicate that carboxymethylation of Cys-282 is responsible for loss of activity. The observations that the activity of the cyanylated glycine methyltransferase shows no decrease upon incubation with iodoacetate and, conversely, the residual activity associated with the iodoacetate-modified enzyme is not abolished by DTNB suggest that Cys-282 is also involved in the inactivation by DTNB. Besides this residue, Cys-185, Cys-246, and Cys-262 are modified upon prolonged incubation with iodoacetate. 5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates glycine methyltransferase by forming 1 disulfide/subunit [Fujioka, M., & Ishiguro, Y. (1986) J. Biol. Chem. 261, 6346-6351]. Despite this stoichiometry, treatment of the FSBA-inactivated enzyme with unlabeled iodoacetate and then with iodo[14C]acetate after reduction with 2-mercaptoethanol and subsequent peptide analysis show that the incorporated radioactivity is distributed equally among Cys-185, Cys-246, Cys-262, and Cys-282.(ABSTRACT TRUNCATED AT 250 WORDS)
Fujioka
M
M
Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
Takata
Y
Y
Konishi
K
K
Ogawa
H
H
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002250:Carbon Radioisotopes; D003545:Cysteine; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D050938:Glycine N-Methyltransferase; D007461:Iodoacetates / Q000494:pharmacology*; D019807:Iodoacetic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000378:metabolism*; D009579:Nitrobenzoates / Q000494:pharmacology*; D010446:Peptide Fragments / Q000032:analysis; D051381:Rats; D014357:Trypsin
D000596:Amino Acids; D002250:Carbon Radioisotopes; D007461:Iodoacetates; D009579:Nitrobenzoates; D010446:Peptide Fragments; D004228:Dithionitrobenzoic Acid; D008780:Methyltransferases; D050938:Glycine N-Methyltransferase; D014357:Trypsin; D003545:Cysteine; D019807:Iodoacetic Acid