The inhibition of glutamate racemase by D-N hydroxyglutamate

Bioorganic and Medicinal Chemistry Letters

Volumn 7, Issue 17, 1997, Pages 2265-2270

  Glavas, Suzana ^a   Tanner, Martin E ^a  

^a UNIVERSITY OF BRITISH COLUMBIA   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

2 OXOGLUTARIC ACID; GLUTAMIC ACID; GLUTAMIC ACID DERIVATIVE; RACEMASE;

ARTICLE; COMPETITIVE INHIBITION; ENANTIOMER; ENZYME INHIBITION; ENZYME KINETICS; ENZYME SUBSTRATE COMPLEX; NONHUMAN;

EID: 0030985410     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0960-894X(97)00413-7     Document Type: Article

References (37)

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26. red, p-iodonitrotetrazolium violet in a reduced form; DAP, diaminopimelate; Trien, triethanolamine
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29. L-N-Hydroxyglutamate (2 mg, 0.012 mmol) was dissolved in deuterated potassium phosphate buffer (250 mM, 0.5 mL, pD 8) containing 0.2 mM dithiothreitol (final pD of approx. 7 by pH indicator paper). Glutamate racemase (125 units in the same buffer) was added and the progress of the reaction was monitored at 37°C using 1H NMR spectroscopy (400 MHz). An analogous experiment with D-N-hydroxyglutamate was run using 45 units of glutamate racemase.
30. -1.
31. 4Cl, 12 units L-glutamate dehydrogenase and 280 units glutamate racemase (1 mL total volume). The reaction was followed to completion and first order rate constants describing the decrease in absorbance at 340 nm were obtained using the program GraFit.
32. D-N-Hydroxyglutamate (1.2 mM) was incubated for 12 min. at 30 °C in 0.45 mL of 50 mM Trien-HCl buffer, pH 8.0, containing 0.02 mM dithiothreitol and 14.5 units of glutamate racemase. A 0.20 mL aliquot of the reaction was quenched by the addition of 3 μL of conc. HCl to a final pH of 2 and the enzyme was removed by ultrafiltration through a Millipore Ultrafree-4 centrifugal filter. To a 50 μL aliquot of the filtrate was added 50 μL of Trien-HCl buffer (0.5 M, pH 8 (final pH of approx. 7 by pH indicator paper)). The sample was assayed for the presence of a racemase inhibitor with 0.3 mM D-glutamate. A control sample lacking racemase was prepared and analyzed in a similar fashion.
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