Function and reactivity of sulfhydryl groups of rat liver glycine methyltransferase

Journal Article · Tue Sep 08 00:00:00 EDT 1987 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00392a018· OSTI ID:5726164

Fujioka, M; Takata, Y; Konishi, K; Ogawa, H

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. Despite this stoichiometry, treatment of the FSBA-inactivated enzyme with unlabeled iodoacetate and then with iodo(/sup 14/C)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.

• Research Organization: Toyama Medical and Pharmaceutical Univ. Faculty of Medicine, Japan
• OSTI ID: 5726164
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:18; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETATES
AMINO ACIDS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL FUNCTIONS
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CYSTEINE
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYME INHIBITORS
ENZYME REACTIVATION
ENZYMES
FUNCTIONS
GLANDS
GLYCINE
HYDROLASES
ISOTOPE APPLICATIONS
KINETICS
LABELLED COMPOUNDS
LIVER
MAMMALS
METHYL TRANSFERASES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PEPTIDE HYDROLASES
PEPTIDES
PROTEINS
RADICALS
RATS
REACTION KINETICS
RODENTS
SERINE PROTEINASES
STOICHIOMETRY
SULFHYDRYL RADICALS
THIOLS
TRACER TECHNIQUES
TRANSFERASES
TRYPSIN
VERTEBRATES