PMID: 1126928May 25, 1975

N-Methylglutamate synthetase. Substrate-flavin hydrogen transfer reactions probed with deazaflavin mononucleotide

The Journal of Biological Chemistry
M S Jorns, L B Hersh


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 methylamin...Continue Reading

Related Concepts

Sodium Dithionite
Fluorescence Spectroscopy
Plasma Protein Binding Capacity
Spectrophotometry, Ultraviolet
Ion-Exchange Chromatography Procedure
Flavin Mononucleotide Monosodium Salt, Dihydrate

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