Role of Residues Related to the Oxidative Reaction at the Aromatic Ring of Substrate in Human 4-Hydroxylphenylpyruvate Dioxygenase

• Main Authors: Yin, Lo, 羅尹
• Other Authors: Hwei-Jen Lee
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/46633795649225353693

碩士 === 國防醫學院 === 生物化學研究所 === 103 === 4-hydroxypheylpyruvate dioxygenase (HPPD) catalyses the second step in tyrosine catabolism involving conversion of 4-hydroxyphenylpyruvate (4-HPP) to homogentisate (HG) via steps of decarboxylation, aromatic hydroxylation and substituent migration. and aromatic hydroxylation. HPPD is a nonheme iron-dependent dioxygenase that have the common 2-His 1-carboxylate facial triad for iron binding. According to the inferred catalytic mechanism, the high reactive O=Fe(IV) intermediate produced after decarboxylation of substrate reacted with aromaric ring of substrate. However, how does it oxidize on the aromatic ring and migrate the side chain so efficiently? What is the realationship with residues in the active site? It remains to be clarified. Hydroxymandelate synthase (HMS) catalyzed the same substrate as HPPD, but the product was S-Hydoxymandelate (HMA). Their sequence shares 25% -35% similarity, but the structures are similar. Till now, only the HMS-HMA complexed structure was resovled. Based on the information, four residues S226, N241, P239, F364 in the active site of HPPD were mutated. Compare to the wild type enzyme, the specific activity, as measured by HG prodution, for S226A, P239T, N241S, F364I and P239T/F364I mutants were decreased by 0.9%, 2.07%, 2.36%, 2.15% and 0.09%, respectively. The specific activity as measured by oxygen comsumed. As compared to wild type, were decreased by 10.57%, 3.61%, 13.66%, 12.89% and 4.38% for S226A, P239T, N241S, F364I and P239T/F364I mutants, respectively. The no consisted of specific activities from two different measurement is because the production of new products. Kinetic analysis showed that the Km values of P239T, N241S and P239T/F364I were increased by 4.6, 4 and 6-fold, respectively, as compared to wild type enzyme. The kcat value of S226A, P239T, N241S, F364I and P239T/F364I were decreased by 4, 19, 5, 5 and 13-fold, respectively, as compared to wild type enzyme. It is noted that substrate inhibition was observed for both S226A and F364I mutants. The behavior could be reduced by raising the concertration of ascorbate. ITC analysis showed the binding of HPP with HPPD/Co2+ complex was exothermic. The caculated thermodynamic parameters for kd, ΔH and ΔS were 6835 µM, 25190 cal/mol and -58.1 cal/mol/deg, respectively. The thermal stability assay by circular dichrorism indicated the Tm values of P239T, F364I and P239T/F364I were 12 ℃, 6 ℃ and 16 ℃ higher than the wild type, respectively. The Tm values of S226A and P239T complex with Co2+ and HPA were 4 ℃ and 2 ℃ higher than their complexes with Co2+, respectively. The result indicated that binary complex of S226A and P239T with Co2+ has high affinity with HPA. The Tm values of N241S complex with Co2+ and HG were 7 ℃ higher than its complex with Co2+. The result indicated that N241S- Co2+ complex has high affinity with HG. Comparing the simulated model of wild type and mutant HPPD, subtitation of these residues affect the stable binding of the aromatic side chain of HPA, which may affect the orientation of Fe(IV)=O for oxidation reaction. Thus the catalytic efficiency of Fe(IV)=O was decreased. The mutation studies and modeled structure indicated the important role of these residues in catalysis.