%0 Journal Article
%T Kinetic Studies of a Coenzyme B12 Dependent Reaction Catalyzed by Glutamate Mutase from <i>Clostridium cochlearium</i>
%A Fredrick Edwin Lyatuu
%A Wolfgang Buckel
%J Advances in Enzyme Research
%P 72-90
%@ 2328-4854
%D 2021
%I Scientific Research Publishing
%R 10.4236/aer.2021.94007
%X <p style=\"margin-left:10.0pt;\">
	<br />
</p>
<p style=\"margin-left:10.0pt;\">
	<span>The
coenzyme B<sub>12</sub> dependent glutamate mutase is composed of two apoenzyme
proteins subunits; S and E<sub>2</sub>, which while either fused or separate
assemble with coenzyme B<sub>12</sub> to form an active holoenzyme (E<sub>2</sub>S<sub>2</sub>-B<sub>12</sub>)
for catalyzing the reversible isomerization between (<i>S</i>)-glutamate and (2<i>S</i>, 3<i>S</i>)-3-methylas</span><span>- </span><span>partate. In order to assay the activity of glutamate
mutase by UV spectrophotometry, this reaction is often coupled with
methylaspartase which deaminates (2<i>S</i>,
3<i>S</i>)-3-methylaspartate to form
mesaconate (<i>¦Ë</i><sub>max</sub> = 240 nm, </span><span>&#400;</span><sub><span>240</span></sub><span> = 3.8 mM<sup>-1</sup>¡¤cm<sup>-1</sup>). The
activities of different reconstitutions of glutamate mu<span>tase from separate apoenzyme components S and E in varied amount</span></span><span>s</span><span> of </span><span>coenzyme B<sub>12</sub> and adenosylpeptide B<sub>12</sub> as cofactors were measured by this assay and used to reveal the binding
properties of the cofactor by the Michaelis</span><span>- </span><span>Menten Method. The values of <i>K<sub>m</sub></i> for coenzyme B<sub>12</sub> in due to reconstitutions
of holoenzyme in 2, 7 and 14 S: E were determined as; 1.12 ¡À 0.04 ¦ÌM, 0.7 ¡À
0.05 ¦ÌM and 0.52 ¡À 0.06 ¦ÌM, respectively, so as those of adenosylpeptide B<sub>12</sub>;
1.07 ¡À 0.04 ¦ÌM and 0.35 ¡À 0.05 ¦ÌM as obtained from respective 2 and 14 S: E
compositions of holoenzyme. Analysis of these kinetics results curiously as<span>sociate</span></span><span>s</span><span> the
increasing affinity of cofactors to apoenzyme with</span><span> </span><span>increased
amount of component S used in reconstituting holoenzyme from separate</span><span> apoenzyme components and cofactor.</span><span> 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 (<i>¦Ë</i><sub>max</sub> = 340 nm, </span><span>&#400;</span><sub><span>340</span></sub><span> = 6.3 mM<sup>-1</sup>¡¤cm<sup>-1</sup>) as determined by UV spectrophotometry
after addition of (2<i>S</i>,<span> 3<i>S</i>)-3-methylaspartate
and pyruvate to a mixture of E<sub>2</sub>S<sub>2</sub>-B<sub>12</sub> and two
auxiliary </span><span>holoenzymes system;
pyridoxal-5-phosphate dependent glutamate-pyruvate </span><span>aminotransferase and N</span>ADH dependent (<i>R</i>)-2-hydroxyglutarate
%K Coenzyme B12
%K Adenosylpeptide B12
%K Glutamate Mutase
%K (S)-Glutamate
%K (2S
%K 3S)-3-Methylaspartate
%K Methylasparatase
%U http://www.scirp.org/journal/PaperInformation.aspx?PaperID=114199