Functional Roles of Tyrosine 91 and Asparagine 442 in the Catalysis of Pigeon Liver Malic Enzyme

• Main Authors: Li-Chen Yen, 顏莉蓁
• Other Authors: Wei-Yuan Chou
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/89856064217978185514

碩士 === 國防醫學院 === 生物化學研究所 === 93 === Pigeon liver malic enzyme catalyzes the reversible oxidative decarboxylation of L-malate to pyruvate and CO2 in the presence of divalent metal ion (Mg+2 or Mn+2), with the concomitant reduction of NADP+ to NADPH. Malic enzyme was proposed to follow a general acid-base catalysis mechanism. The crystal structure of pigeon malic enzyme shows that Tyr91 and Lys162 are likely candidates for the catalytic residues. Several other highly conserved residues, including Asn442, are part of the hydrogen bonding network in the active site. Therefore, Asn442 may have important roles in the catalysis. In this study, Asn442 was mutated to alanine, aspartic acid and glutamine. The catalytic activities for N442A and N442D mutants were completely lost, and the kcat value for N442Q is 5000 fold lower than that of wild type. For partial reaction studies, N442A and N442D demonstrated wild type enzyme activity for pyruvate reduction reaction, but no detectable oxalacetate decarboxylation activity. From these results, Asn442 is assumed to involve in the step of tautomerization. For N442A and N442D, addition of ammonium chloride (10 mM) exhibits substantial, yet incomplete, restoration of the overall reaction and decarboxylation activities. From the 3D-structure, Tyr91 has been proposed to act as the general acid (Biochemistry, Vol.42, 12721-12733, 2003). To further examine the relationship of Tyr91 and Asn442, double mutants of Y91F/N442A, Y91F/N442D and Y91F/N442Q were constructed. They also have no detectable enzymatic activities. Addition of ammonium chloride (10 mM) exhibits 70～80 folds restoration in activities. In conclusion, Tyr91 and Asn442 may form a catalytic diad to facilitate the proton transfer in enolpyruvate-pyruvate tautomerization.