Conserved arginine-516 of Penicillium amagasakiense glucose oxidase is essential for the efficient binding of beta-D-glucose.
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 interaction between 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.
Witt
S
S
GBF (Gesellschaft für Biotechnologische Forschung), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Wohlfahrt
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G
Schomburg
D
D
Hecht
H J
HJ
Kalisz
H M
HM
D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D002942:Circular Dichroism; D017124:Conserved Sequence / Q000235:genetics*; D004926:Escherichia coli; D005947:Glucose / Q000378:metabolism*; D005949:Glucose Oxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D011994:Recombinant Proteins; D001120:Arginine; D005949:Glucose Oxidase; D005947:Glucose