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Titolo:
Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose
Autore:
Witt, S; Wohlfahrt, G; Schomburg, D; Hecht, HJ; Kalisz, HM;
Indirizzi:
Gesell Biotechnol Forsch GmbH, D-38124 Braunschweig, Germany Gesell Biotechnol Forsch GmbH Braunschweig Germany D-38124 weig, Germany
Titolo Testata:
BIOCHEMICAL JOURNAL
, volume: 347, anno: 2000,
parte:, 2
pagine: 553 - 559
SICI:
0264-6021(20000415)347:<553:CAOPAG>2.0.ZU;2-O
Fonte:
ISI
Lingua:
ENG
Soggetto:
ASPERGILLUS-NIGER; GMC OXIDOREDUCTASES; ANGSTROM RESOLUTION; PROTEINS; PROGRAM;
Keywords:
active site; kinetic studies; molecular modelling; site-directed mutagenesis;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
34
Recensione:
Indirizzi per estratti:
Indirizzo: Kalisz, HM Gesell Biotechnol Forsch GmbH, Mascheroder Weg 1, D-38124 Braunschweig, Germany Gesell Biotechnol Forsch GmbH Mascheroder Weg 1 Braunschweig Germany D-38124
Citazione:
S. Witt et al., "Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose", BIOCHEM J, 347, 2000, pp. 553-559

Abstract

The effects of mutation of key conserved active-site residues (Tyr-73, Phe-418, Trp-430, Arg-516, Asn-518, His-520 and His-563) of glucose oxidase from Penicillium amagasakiense on substrate binding were investigated. Kinetic studies on the oxidation of beta-D-glucose combined with molecular modelling showed the side chain of Arg-516, which forms two hydrogen bonds with the 3-OH group of beta-D-glucose, to be absolutely essential for the efficient binding of beta-D-glucose. The R516K variant, whose side chain forms only one hydrogen bond with the 3-OH group of beta-D-glucose, exhibits an 80-fold higher apparent K-m (513 mM) but a V-max only 70% lower (280 units/mg) than the wild type. The complete elimination of a hydrogen-bond interactionbetween residue 516 and the 3-OH group of beta-D-glucose through the substitution R516Q effected a 120-fold increase in the apparent K-m for glucose (to 733 mM) and a decrease in the V-max to 1/30 (33 units/mg). None of the other substitutions, with the exception of variant F418A, affected the apparent K-m more than 6-fold. In contrast, the removal of aromatic or bulky residues at positions 73, 418 or 430 resulted in decreases in the maximum rates of glucose oxidation to less than 1/90. Variants of the potentially catalytically active His-520 and His-563 were completely, or almost completely,inactive. Thus, of the residues forming the active site of glucose oxidase, Arg-516 is the most critical amino acid for the efficient binding of beta-D-glucose by the enzyme, whereas aromatic residues at positions 73, 418 and 430 are important for the correct orientation and maximal velocity of glucose oxidation.

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Documento generato il 30/11/20 alle ore 19:51:09