Recombinant rat liver guanidinoacetate methyltransferase: Reactivity and function of sulfhydryl groups

Journal Article · Tue Oct 04 00:00:00 EDT 1988 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00420a013· OSTI ID:7118386

Fujioka, Motoji; Konishi, Kiyoshi; Takata, Yoshimi ^[1]

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1. Toyama Medical and Pharmaceutical Univ. (Japan)

Rat liver guanidinoacetate methyltransferase, produced in Escherichia coli by recombinant DNA technique, possesses five cysteine residues per molecule. No disulfide bond is present. Analysis of the chymotryptic peptides derived from the iodo({sup 14}C)acetate-modified enzyme shows that Cys-90, Cys-15, Cys-219, and Cys-207 are alkylated by the reagent in order of decreasing reactivity. Incubation of the enzyme with excess 5,5{prime}-dithiobis(2-nitrobenzoate) (DTNB) in the absence and presence of cystamine (2,2{prime}-dithiobis(ethylamine)) causes the appearance of 4 and 5 mol of 2-nitro-5-mercaptobenzoate/mol of enzyme, respectively. Reaction of the methyltransferase with an equimolar amount of DTNB results in an almost quantitative disulfide cross-linking of Cys-15 and Cys-90 with loss of a large portion of the activity. The methyltransferase is completely inactivated by iodoacetate following nonlinear kinetics. Comparison of the extent of inactivation with that of modification of cysteine residues and the experiment with the enzyme whose Cys-15 and Cys-90 are cross-linked suggest that alkylation of Cys-15 and Cys-90 results in a partially active enzyme and that carboxymethylation of Cys-219 completely eliminates enzyme activity. The inactivation of guanidinoacetate methyltransferase by iodoacetate or DTNB is not protected by substrates. Furthermore, disulfide cross-linking of Cys-15 and Cys-90 or carboxymethylation of Cys-219 does not impair the enzyme's capacity to bind S-adenosylmethionine. Thus, these cysteine residues appear to occur outside the active-site region, but their integrity is crucial for the expression of enzyme activity.

• OSTI ID: 7118386
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 27:20; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETATES
ALKYLATION
AMINO ACID SEQUENCE
AMINO ACIDS
ANIMALS
BACTERIA
BODY
CARBON 14 COMPOUNDS
CARBOXYLIC ACID SALTS
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
CHROMATOGRAPHY
CYSTEINE
DIGESTIVE SYSTEM
DNA
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
GLANDS
LABELLED COMPOUNDS
LIQUID COLUMN CHROMATOGRAPHY
LIVER
MAMMALS
METHYL TRANSFERASES
MICROORGANISMS
MOLECULAR STRUCTURE
NITRO COMPOUNDS
NUCLEIC ACIDS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
RATS
RECOMBINANT DNA
RODENTS
SEPARATION PROCESSES
SUBSTRATES
THIOLS
TRANSFERASES
VERTEBRATES