<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...<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>λ</i><sub>max</sub> = 240 nm, </span><span>Ɛ</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 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B<sub>12</sub>;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 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>λ</i><sub>max</sub> = 340 nm, </span><span>Ɛ</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 dehydrogenas<span>e. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (<i>S</i>)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (<i>R</i>)-2-hydroxylglutarate</span> dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2<i>S</i>, 3<i>S</i>)-3-methylaspartate in the reaction of glutamate mutase as <i>K</i><sub>m</sub>= 7 ± 0.07 mM and <i>k</i><sub>cat</sub>= 0.54 ± 0.6 s<sup>-1</sup>. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, <i>K</i><sub>eq</sub> = [(<i>S</i>)-glutamate] × [(2<i>S</i>, 3<i>S</i>)-3-methylaspartate]<sup>-1</sup> = 16, where the kinetic constants of (<i>S</i>)-glutamate were determined by the standard methylaspartase coupled assay.<span></span></span> </p> <p> <br /> </p>展开更多
文摘<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>λ</i><sub>max</sub> = 240 nm, </span><span>Ɛ</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 ± 0.04 μM, 0.7 ± 0.05 μM and 0.52 ± 0.06 μM, respectively, so as those of adenosylpeptide B<sub>12</sub>;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 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>λ</i><sub>max</sub> = 340 nm, </span><span>Ɛ</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 dehydrogenas<span>e. The activity of glutamate-pyruvate aminotransferase was relatively complete recovered upon the addition of (<i>S</i>)-glutamate and pyruvate to the mixtures of hologlutamate-pyruvate aminotransferase and (<i>R</i>)-2-hydroxylglutarate</span> dehydrogenase which were incubated with each putative inhibitor of glutamate mutase. Additionally, the new assay was used to determine the kinetic constants of (2<i>S</i>, 3<i>S</i>)-3-methylaspartate in the reaction of glutamate mutase as <i>K</i><sub>m</sub>= 7 ± 0.07 mM and <i>k</i><sub>cat</sub>= 0.54 ± 0.6 s<sup>-1</sup>. Application of Briggs-Haldane formula allowed the calculation of an equilibrium constant of the reversible isomerization, <i>K</i><sub>eq</sub> = [(<i>S</i>)-glutamate] × [(2<i>S</i>, 3<i>S</i>)-3-methylaspartate]<sup>-1</sup> = 16, where the kinetic constants of (<i>S</i>)-glutamate were determined by the standard methylaspartase coupled assay.<span></span></span> </p> <p> <br /> </p>
