title: Rat liver guanidinoacetate methyltransferase. Proximity of cysteine residues at positions 15, 90 and 219 as revealed by site-directed mutagenesis and chemical modification.
abstract: Cys-90 of rat liver guanidinoacetate methyltransferase is a very reactive residue, and chemical modification of this residue results in a large decrease in activity [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. To understand better the role of Cys-90 in catalysis, this residue was replaced with alanine by oligonucleotide-directed mutagenesis. The mutant is active and has kinetic constants similar to those of wild-type, indicating that Cys-90 is not involved in catalysis and substrate binding. The u.v.-absorption, fluorescence and c.d. spectra are also unchanged. Reaction of the mutant with an equimolar amount of 5,5'-dithiobis-(2-nitrobenzoic acid) or 2-nitro-5-thiocyanobenzoic acid results in an almost quantitative disulphide cross-linking between Cys-15 and Cys-21). The same treatment effects disulphide bond formation between Cys-15 and Cys-90 in wild type [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. Since the mutant and wild-type enzymes appear to have similar secondary and tertiary structures, these results suggest that Cys-15, Cys-90 and Cys-219 of the methyltransferase occur spatially close together. The mutant cross-linked between Cys-15 and Cys-219 and the wild-type cross-linked between Cys-15 and Cys-90 show very similar spectroscopic properties. Although treatment of the mutant and wild-type enzymes with equimolar concentrations of 5,5'dithiobis-(2-nitrobenzoic acid) causes a large loss of enzyme activity in each case, kinetic analyses with the modified enzymes suggest that cross-linking of Cys-15 with Cys-90 or Cys-219 does not abolish activity and does not result in a large change in the Michaelis constants. Incubation of the mutant enzyme with excess 2-nitro-5-thiocyanobenzoic acid leads to modification of Cys-207 in addition to Cys-15 and Cys-219. Retention of considerable enzyme activity in the modified enzyme indicates that Cys-207 is also not an essential residue.
authors.affiliation: Department of Biochemistry, Toyama Medical and Pharmaceutical University Faculty of Medicine, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Takata
authors.forename: T
authors.initials: T
authors.lastname: Date
authors.forename: M
authors.initials: M
authors.lastname: Fujioka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003545:Cysteine; D004220:Disulfides / Q000032:analysis; D050937:Guanidinoacetate N-Methyltransferase; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D011487:Protein Conformation; D051381:Rats
chemical_list: D004220:Disulfides; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D008780:Methyltransferases; C494508:Gamt protein, rat; D050937:Guanidinoacetate N-Methyltransferase; D003545:Cysteine