| Summary: | 碩士 === 國防醫學院 === 生物化學研究所 === 88 === Pigeon liver malic enzyme [EC1.1.1.40] is a homotetramer with each subunit of molecular weight 65 kDa. This enzyme is a bifunctional enzyme that catalyzes the reversible oxidative decarboxylation of L-malate, yielding pyruvate and CO2, with a concomitant conversion of coenzyme NADP+ to NADPH. Previous experiments in our laboratory found that when glutamate and lysine at position 9 and 17 of pigeon liver malic enzyme were replaced by arginine and methionine, respectively, the enzyme became much more stable against urea and thermal denaturation, but was enzymatically less active than the wild-type enzyme (WT). The double mutant R9E/M17K, however, is less stable than the wild type in urea denaturation in the presence of high concentration of NaCl. Coupling energy, △△Gint, in the double mutant indicates a strong interaction between E9 and K17. These results imply that the increased stability gained may be due to an extra ion-pair between E9 and K17. To complete analysis of this possible ion-pair interaction, alanine was used at the mutation sites to avoid new interactions being formed. More information should be available by analyzing the interactions of the residues directly. The R9E/M17K 'not-to-alanine' double-mutant cycles was then decomposed into four double-mutant cycles with mutations to alanine at either or both of the sites. It revealed that the coupling energies of these four mutation cycles are equal to that of 'not-to-alanine' double-mutant cycle. The half unfolding urea concentration values estimated by fluorescence spectral, fluorescence intensity and circular dichroism signal changes were all increased for the double mutant (R9E/M17K). In addition, strong interaction between E9 and K17 can be observed in the coupling energy for R9A/M17A, R9A/M17K, R9E/M17A and R9/AM17K only in fluorescence spectral shift experiment. Structure evidence is needed to confirm the extra ion pair. The study of pigeon malic enzyme quaternary structure may be able to get some evidences to explain the stability of R9E/M17K.
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