| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 100 === 4-hydroxyphenylpyruvate dioxygenase(4-HPPD; EC 1.13.11.27) catalyzes the second step in the pathway for the catabolism of tyrosine.
It incorporates both atoms of oxygen into 4-hydroxyphenylpyruvate to form homogentisate, a reaction that involves decarboxylation, substituent migration , and aromatic oxygenation. 4-HPPD belongs to the alpha-keto acid-dependent subgroup of non-heme dioxygenases. A ferrous iron binds in the active site in a way of 2-His-1-carboxylate facial triad. Alignments of amino acid sequence and crystal structure confirmed that H183, H266 and E349 coordinated with ferrous ion in active site. To understand the role of three amino acid site-directed mutagenesis was investigated. Wild-type and mutant 4-HPPD have similar secondary structure as analysed by CD. The specific activities of wild-type HPPD was determined to be 3±0.5 nmole/min/μg and 3.2±0.2 nmole/min/μg of by oxygraph and HPLC assay, respectively. The specific activity of mutant of H183A、H266A、E349D、E349G、E349Q have about 2.1%、10.7%、19.3%、2.7%、17.7% , Mutant of H183A、E349D、E349G compared with wild-type have about 0.64%、16.4%、2.6% by oxygraph and hplc assay, respectively. The HPP kinetic analysis showed increased Km value for E349Q.The kcat and kcat /Km were significantly decreased as compared to these of wild-type enzyme. The Km value of Fe2+ for H266A and E349Q were 21-fold and 12- fold increased .The kcat and kcat /Km were significant decreased as compared to wild-type enzyme. In addition , HPA product were detected for H266A indicate the uncouple rex. This study indicates the crucial and distinct role of the three amino acids in ferrous binding and catalysis.
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