The coenzyme B₁₂ dependent glutamate mutase is composed of two apoenzyme proteins subunits; S and E₂, which while either fused or separate assemble with coenzyme B₁₂ to form an active holoenzyme (E₂S₂-B₁₂) for catalyzing the reversible isomerization between (S)-glutamate and (2S, 3S)-3-methylas- partate. In order to assay the activity of glutamate mutase by UV spectrophotometry, this reaction is often coupled with methylaspartase which deaminates (2S, 3S)-3-methylaspartate to form mesaconate (λ_max = 240 nm, Ɛ₂₄₀ = 3.8 mM^-1·cm^-1). The activities of different reconstitutions of glutamate mutase from separate apoenzyme components S and E in varied amounts of coenzyme B₁₂ and adenosylpeptide B₁₂ as cofactors were measured by this assay and used to reveal the binding properties of the cofactor by the Michaelis- Menten Method. The values of K_m for coenzyme B₁₂ in due to reconstitutions of holoenzyme in 2, 7 and 14 S: E were determined as; 1.12 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B₁₂; 1.07 ± 0.04 μM and 0.35 ± 0.05 μM as obtained from respective 2 and 14 S: E compositions of holoenzyme. Analysis of these kinetics results curiously associates the increasing affinity of cofactors to apoenzyme with increased amount of component S used in reconstituting holoenzyme from separate apoenzyme components and cofactor. Moreover, in these studies a new method for assaying the activity of glutamate mutase was developed, whereby glutamate mutase activity is measured via depletion of NADH (λ_max = 340 nm, Ɛ₃₄₀ = 6.3 mM^-1·cm^-1) as determined by UV spectrophotometry after addition of (2S, 3S)-3-methylaspartate and pyruvate to a mixture of E₂S₂-B₁₂ and two auxiliary holoenzymes system; pyridoxal-5-phosphate dependent glutamate-pyruvate aminotransferase and NADH dependent (R)-2-hydroxyglutarate dehydrogenase. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (S)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (R)-2-hydroxylglutarate dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2S, 3S)-3-methylaspartate in the reaction of glutamate mutase as K_m= 7 ± 0.07 mM and k_cat= 0.54 ± 0.6 s^-1. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, K_eq = [(S)-glutamate] × [(2S, 3S)-3-methylaspartate]^-1 = 16, where the kinetic constants of (S)-glutamate were determined by the standard methylaspartase coupled assay.