N-Methylglutamate synthetase. Substrate-flavin hydrogen transfer reactions probed with deazaflavin mononucleotide.
N-Methylglutamate synthetase, reconstituted from apo-protein with 5-deazaFMN, catalyzes the reversible formation of N-methylglutamate via the same two-step mechanism previously elucidated for native enzyme (Reactions 1 and 2).(See article). This conclusion is based on the observation that: 1. Enzyme-bound deazaFMN (gamma-max equals 410, 338, epsilon410 equals 10,400 m-minus 1 cm-minus 1) is reduced by L-glutamate, N-methyl-L-glutamate but not D-glutamate. At saturating concentrations of L-glutamate Reaction 1 proceeds at 1% of the rate observed with FMN-reconstituted enzyme. 2. Substrate-reduced deazaFMN enzyme is reoxidized by methylamine or ammonia. 3. A glutaryl enzyme intermediate, isolated by Sephadex G-25 chromatography, contains radioactivity when prepared from [U-14C]glutamate, [alpha-3H]glutamate, or N-[glutaryl U-14C]methylglutamate; however, this intermediate is not labeled from N[methyl 14C]methylglutamate. 4. The amount of radioactivity incorporated into the intermediate is stoichiometric with the amount of deazaFMN reduced during its formation. 5. Intermediate prepared with [U-14C]glutamate yields alpha-[14C]ketoglutarate when denatured with acid and N-[glutaryl-U-14C]methylglutamate when incubated with methylamine. In the absence of methylamine deazaFMN enzyme intermediate slowly decays to yield alpha-hydroxyglutarate. 6. The rate of deazaFMN glutaryl enzyme intermediate formation at a fixed glutamate concentration is equal to the rate of the over-all reaction while the rate of intermediate reaction with methylamine is approximately 50 times greater than the over-all reaction. DeazaFMN enzyme intermediate prepared with [alpha-3H]-glutamate yields [3H]deazaFMNH2 when denatured with acid or phenol and N-[3H]methylglutamate when incubated with methylamine. These results show that the alpha-hydrogen of glutamate is transferred to deazaFMNH2, presumably at the 5 position, during Reaction 1 and that the same hydrogen is utilized for the reformation of the alpha C-H bond during Reaction 2. These results provide the first direct evidence for enzymic hydrogen transfer from substrate to flavin.
D001051:Apoenzymes; D001665:Binding Sites; D002852:Chromatography, Ion Exchange; D004227:Dithionite; D005486:Flavin Mononucleotide / Q000031:analogs & derivatives*; D005971:Glutamates; D007700:Kinetics; D008744:Methylamines; D008780:Methyltransferases / Q000378:metabolism*; D010084:Oxidation-Reduction; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
D001051:Apoenzymes; D005971:Glutamates; D008744:Methylamines; D004227:Dithionite; D005486:Flavin Mononucleotide; D008780:Methyltransferases