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Bulletin of the Korean Chemical Society (BKCS)

ISSN 0253-2964(Print)
ISSN 1229-5949(Online)
Volume 8, Number 4
BKCSDE 8(4)
April 20, 1987

Title	Chemical Modification of Glycolate Oxidase from Spinach by Diethyl Pyrocarbonate. Evidence of Essential Histidine for Enzyme Activity
Author	Kun Kook Lee, Hong Sun Kim, Jung Do Choi*
Keywords
Abstract	FMN-dependent glycolate oxidase from spinach is inactivated by diethyl pyrocarbonate at pH 7.0. Inactivation of both apo- and holoenzyme by diethyl pyrocarbonate follows pseudo-first-order kinetics and first order with respect to the reagent. A series of difference spectra of inactivated and native enzymes show a single peak at 240 nm, indicating the modification of histidyl residues. No decrease in absorbance at around 280 nm due to formation of O-carbethoxytyrosine is observed. The rate of inactivation is dependent on pH, and the data for pH dependent rates implicate the involvement of a group with a pKa of 6.9. The activity lost by treatment with diethyl pyrocarbonate could be almost fully restored by incubation with 0.75M hydroxylamine. The reactivation by hydroxylamine and the pH dependence of inactivation are also consistent with that the inactivation is due to modification of histidyl residues. Although coenzyme FMN is without protective effect, the substrate glycolate,the product glyoxylate, and two competitive inhibitors, oxalate and oxalacetate, provide marked protection against the inactivation of the holoenzyme. These results suggest that the inactivation of the oxidase by diethyl pyrocarbonate occurs by modification of essential histidyl residue(s) at the active site.
Page	280 - 285
Full Text	PDF

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