Witt S, Wohlfahrt G, Schomburg D, Hecht H J, Kalisz H M
GBF (Gesellschaft für Biotechnologische Forschung), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
Biochem J. 2000 Apr 15;347(Pt 2):553-9. doi: 10.1042/0264-6021:3470553.
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.
研究了天草青霉葡萄糖氧化酶关键保守活性位点残基（Tyr-73、Phe-418、Trp-430、Arg-516、Asn-518、His-520和His-563）突变对底物结合的影响。对β-D-葡萄糖氧化的动力学研究结合分子模拟表明，与β-D-葡萄糖的3-OH基团形成两个氢键的Arg-516侧链对于β-D-葡萄糖的有效结合绝对至关重要。R516K变体的侧链仅与β-D-葡萄糖的3-OH基团形成一个氢键，其表观K(m)比野生型高80倍（513 mM），但V(max)仅低70%（280单位/毫克）。通过R516Q取代完全消除残基516与β-D-葡萄糖的3-OH基团之间的氢键相互作用，使葡萄糖的表观K(m)增加了120倍（达到733 mM），V(max)降低至1/30（33单位/毫克）。除了F418A变体之外，其他取代对表观K(m)的影响均不超过6倍。相比之下，在73、418或430位去除芳香族或大体积残基会导致葡萄糖氧化的最大速率降低至不到1/90。潜在催化活性的His-520和His-563的变体完全或几乎完全无活性。因此，在构成葡萄糖氧化酶活性位点的残基中，Arg-516是该酶有效结合β-D-葡萄糖最关键的氨基酸，而73、418和430位的芳香族残基对于葡萄糖氧化的正确取向和最大速度很重要。
