Mechanistic and stereochemical studies on methylaspartase and glutamate mutase
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Preliminary studies have been undertaken on the enzyme, glutamate mutase. Stereochemically pure (2S,3S)-3-ethylaspartic acid has been synthesized. The turnover of this substrate analogue by glutamate mutase has been investigated. Possible reaction products have been synthesized. (2S,3S)-[1',1',2',2',2'-2H]-3-Ethylaspartic acid has been prepared and a novel synthesis of [1-2H]-ethanol has been investigated with the aim of preparing (2S,3S)-[1'-2H]-3-ethylaspartic acid. In order to investigate the mechanism of elimination of ammonia from (2S,3R)-3-methylaspartic acid, by the enzyme 3-methylaspartase, stereospecific routes to (2S,3R)-3-methylaspartic acid and [3-2H]-(2S,3R)-3-methylaspartic acid have been explored. The compounds were obtained in high enantiomeric excess and with >97 % incorporation of deuterium into the latter. It has been demonstrated that 3-methylaspartase catalyses the direct elimination of ammonia from these substrates, presumably by a syn- elimination mechanism. The kinetic parameters, Vmax and Km, have been determined for both compounds at 1 and 50 mM potassium ion concentrations. A deuterium isotope effect on (D(V)) of 7.15 +/- 2.74 was measured for the reaction at 1 mM potassium ion concentration. A large D(V) of 6.79 + 0.92 was also observed at 50 mM potassium ion concentration, in contrast to results with the natural isomer which show the effect is completely suppressed at this potassium ion concentration. Values for D(V/K) were also obtained at 1 and 50 mM potassium ion concentrations. They were 3.39 +/- 1.6 and 4.10 +/- 1.3, respectively. The 15N isotope effect on V/K was measured at 1 mM potassium ion concentration. A value of 1.0028 +/- 0.0040 was observed for (2S,3R)-3-methylaspartic acid, and, a value of 1.0033 +/- 0.0043 observed for [3-2H]-(2S,3R)-3-methylaspartic acid.
Thesis, PhD Doctor of Philosophy
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