The Search for the active site configuration of glutamate dehydrogenase i) Reactivity of LYS-126 ii) Preparation of O-Se-NADP+

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Title: The Search for the active site configuration of glutamate dehydrogenase i) Reactivity of LYS-126 ii) Preparation of O-Se-NADP+
Author: Judd, Deborah
Abstract: The reactivity of lys-126 in glutamate dehydrogenase was investigated by monitoring the loss of enzymatic activity when lys-126 is modified by pyridoxyl 5'phosphate. The effect of various ligands on the rate constant of inactivation (k inact) was determined. The purine nucleotides ADP and GTP increased the k inact, glutarate slightly increased the k inact , 2-oxoglutarate and succinate were determined to have no effect on k inact and no conclusions could be drawn on L-2- hydroxyglutarate. After the modification of glutamate dehydrogenase with pyridoxyl 5'phosphate, the percent residual activity (%R.A.) was determined and evaluated. ADP, GTP and succiante had no effect on the %R. A, glutarate and 2-oxoglutarate significantly increased the %R.A. No conclusions could be drawn about the effect of L-2-hydroxyglutarate. Modification of lys-126 by pyridoxyl 5'phosphate is reversible, so the effects of the various ligands on the rate constant of reactivation (k react) was also investigated. Both the purine nucleotides, ADP and GTP, as well as the substrate analogs, glutarate, succinate, L-2- hydroxyglutarate,and 2-oxoglutarate, had no effect on the rate constant of reactivation. Using ion exchange chromatography, a protocol for separating NAD + from NADP+ was determined. This separation is a necessary step for the enzymatic synthesis of a selenium NADP+ analog for X-ray crystallography.
Record URI: http://hdl.handle.net/1850/10689
Date: 1991-08

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