| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 107 === 4-Hydroxyphenylpyruvate dioxygenase (4-HPPD) is a nonheme iron/α-oxoacid-dependent dioxygenase that have the common 2-His 1-carboxylate facial triad for iron binding. 4-HPPD catalyses the second step in tyrosine catabolism involving conversion of 4-hydroxyphenylpyruvate (4-HPP) to homogentisate (HG) via steps of decarboxylation, substituent migration and aromatic hydroxylation. We investigated the effect of F359L, F364I, F368Y and F364I/F368Y mutants on the substrate binding and catalytic activity of 4-HPPD. The activity was analyzed using the Oxygraph assay and HPLC to determine the oxygen consumption and HG product formation in the reaction, respectively. It found that the specific activity of the mutant F359L, F364I and F364I/F368Y was reduced by 6.5-, 1.6- and 6.5-fold, respectively, F368Y was increased by 2-fold, as compared with that of the wild type. And the mutant F368Y is similar to the wildtype. Compared with wild type, the Km values for F359L, F364I and F364I/F368Y were reduced by 8-, 6- and 4-fold, respectively. And the mutant F368Y is similar to the wildtype. The kcat/Km values of F359L and F364I/F368Y are similar to that of the wild type, however, F364I and F364I/F368Y was increased by 3-, 2-fold. Substrate inhibition were observed in kinetic analysis for the three mutants. Isothermal titration calorimetry analysis showed that the binding of 4-HPP by 4-HPPD-Co2+ complex was an exothermic reaction. The Kd value of F364I/F368Y was increased by 2.6-fold, F359L, F364I and F368Y was reduced by 2-, 5- and 3-fold, as compared with that of the wild type. The results suggested that the mutants of F359L, F364I, F368Y and F364I/F368Y affect the binding of substrate, in particular the π－π interactions between the aromatic sidechain of substrate and these residues. It might cause the substrate inhibition behavior from reduced substrate turnover rate and Km values in the catalytic reaction of the mutant enzymes.
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