Stenberg K, Clausen T, Lindqvist Y, Macheroux P
Department of Molecular Biology, Swedish University of Agricultural Sciences, Uppsala Biomedical Center.
Eur J Biochem. 1995 Mar 1;228(2):408-16.
Tyr24 and Trp108 are located in the active site of spinach glycolate oxidase. To elucidate their function in substrate binding and catalysis, they were replaced by phenylalanine and serine, respectively. The [Y24F]glycolate oxidase mutant enzyme showed a tenfold higher Km value for glycolate. L-lactate and DL-2-hydroxybutyrate also showed higher Km values, however, the substrate specificity was unchanged as compared to the wild-type enzyme (Km increases in the order glycolate < DL-2-hydroxybutyrate < L-lactate < L-mandelate). The turnover number and the rate of reduction, found to be rate limiting in catalysis, were only slightly affected by the deletion of the hydroxyl group. These findings suggest that Tyr24 is mostly involved in substrate binding. The spectral features of the [Y24F]glycolate oxidase suggest that a fraction (50-80%) of the protein bears a flavin N(5) adduct instead of the oxidized cofactor. Crystals obtained from the isolated [Y24F]glycolate oxidase mutant protein allowed the determination of the three-dimensional structure. Although the structure was low resolution (0.3 nm), it is evident that the structure determined is that of the N(5) adduct species. In addition to the lacking hydroxyl group of Tyr24, we also observed movements of the amino acid side chains of Arg164 and Trp108, the latter replacing a water molecule in the substrate-binding pocket. Other features predominantly found in the class of flavoprotein oxidases, such as stabilization of the covalent N(5)-sulfite adduct and of the paraquinoid form of 8-mercapto-FMN, were found to be conserved. [W108S]Glycolate oxidase, in contrast, showed dramatic effects on both the Km of substrates as well as the turnover number. The Km for glycolate was increased some hundred fold and the turnover number was decreased 500-fold. In addition, it was found that the higher homologs of glycolate, L-lactate and DL-2-hydroxybutyrate had turnover numbers similar to those found with the wild-type enzyme, although the Km values also increased dramatically. These results indicate that Trp108 is of major importance in catalysis and that this residue is involved in determining the substrate specificity of glycolate oxidase.
酪氨酸24（Tyr24）和色氨酸108（Trp108）位于菠菜乙醇酸氧化酶的活性位点。为阐明它们在底物结合和催化中的功能，分别用苯丙氨酸和丝氨酸将它们替换。[Y24F]乙醇酸氧化酶突变体酶对乙醇酸的Km值高出10倍。L-乳酸和DL-2-羟基丁酸也显示出较高的Km值，然而，与野生型酶相比，底物特异性未变（Km按乙醇酸＜DL-2-羟基丁酸＜L-乳酸＜L-扁桃酸的顺序增加）。催化中发现的限速步骤——周转数和还原速率，仅受到羟基缺失的轻微影响。这些发现表明Tyr24主要参与底物结合。[Y24F]乙醇酸氧化酶的光谱特征表明，一部分（50 - 80%）蛋白质带有黄素N(5)加合物而非氧化型辅因子。从分离的[Y24F]乙醇酸氧化酶突变体蛋白获得的晶体使得能够确定其三维结构。尽管该结构分辨率较低（0.3纳米），但很明显所确定的结构是N(5)加合物形式的结构。除了Tyr24缺少羟基外，我们还观察到精氨酸164（Arg164）和色氨酸108的氨基酸侧链发生移动，后者取代了底物结合口袋中的一个水分子。在黄素蛋白氧化酶类别中主要发现的其他特征，如共价N(5)-亚硫酸盐加合物和8-巯基-FMN的对醌形式的稳定化，被发现是保守的。相比之下，[W108S]乙醇酸氧化酶对底物的Km值和周转数都有显著影响。乙醇酸的Km值增加了约100倍，周转数降低了500倍。此外，还发现乙醇酸、L-乳酸和DL-2-羟基丁酸的较高同系物的周转数与野生型酶的相似，尽管它们的Km值也大幅增加。这些结果表明Trp108在催化中至关重要，并且该残基参与确定乙醇酸氧化酶的底物特异性。
