INVOLVEMENT OF TYR24 AND TRP108 IN SUBSTRATE-BINDING AND SUBSTRATE-SPECIFICITY OF GLYCOLATE OXIDASE

Tyr24 and Trp108 are located in the active site of spinach glycolate o xidase. To elucidate their function in substrate binding and catalysis , they were replaced by phenylalanine and serine, respectively. The [Y 24F]glycolate oxidase mutant enzyme showed a tenfold higher K-m value for glycolate. L-lactate and DL-2-hydroxybutyrate also showed higher K -m values, however, the substrate specificity was unchanged as compare d to the wild-type enzyme (K-m increases in the order glycolate < DL-2 -hydroxybutyrate < L-lactate < L-mandelate). The turnover number and t he rate of reduction, found to be rate limiting in catalysis, were onl y slightly affected by the deletion of the hydroxyl group. These findi ngs suggest that Tyr24 is mostly involved in substrate binding. The sp ectral features of the [Y24F]glycolate oxidase suggest that a fraction (50-80%) of the protein bears a flavin N(5) adduct instead of the oxi dized cofactor. Crystals obtained from the isolated [Y24F]glycolate ox idase mutant protein allowed the determination of the three-dimensiona l structure. Although the structure was low resolution (0.3 nm), it is evident that the structure determined is that of the N(5) adduct spec ies. In addition to the lacking hydroxyl group of Tyr24, we also obser ved movements of the amino acid side chains of Arg164 and Trp108, the latter replacing a water molecule in the substrate-binding pocket. Oth er 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 K -m of substrates as well as on the turnover number. The K-m for glycol ate was increased some hundred fold and the turnover number was decrea sed 500-fold. in addition, it was found that the higher homologs of gl ycolate, L-lactate and DL-2-hydroxybutyrate had turnover numbers simil ar to those found with the wild-type enzyme, although the K-m values a lso increased dramatically. These results indicate that Trp108 is of m ajor importance in catalysis and that this residue is involved in dete rmining the substrate specificity of glycolate oxidase.