TITLE:
Kinetic Studies of a Coenzyme B12 Dependent Reaction Catalyzed by Glutamate Mutase from Clostridium cochlearium
AUTHORS:
Fredrick Edwin Lyatuu, Wolfgang Buckel
KEYWORDS:
Coenzyme B12, Adenosylpeptide B12, Glutamate Mutase, (S)-Glutamate, (2S, 3S)-3-Methylaspartate, Methylasparatase
JOURNAL NAME:
Advances in Enzyme Research,
Vol.9 No.4,
December
28,
2021
ABSTRACT:
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 (λmax = 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 Km 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 (λmax = 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 Km= 7 ± 0.07 mM and kcat= 0.54 ± 0.6 s-1.
Application of Briggs-Haldane formula allowed the calculation of an equilibrium
constant of the reversible isomerization, Keq = [(S)-glutamate] × [(2S, 3S)-3-methylaspartate]-1 = 16, where the kinetic constants of (S)-glutamate were determined by
the standard methylaspartase coupled assay.