NMR Studies of the Conformation of E. coli Dihydrofolate Reductase Complexes and the Kinetics of Isotope Exchange Reactions of Malonic Acids

• Main Authors: Hsu Ming-Ching, 許銘清
• Other Authors: Huang Fu-Yung
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/80257507551268279534

博士 === 國立成功大學 === 化學系 === 87 === Abstract Multiple 13C-labelled methotrexate (MTX), [2,N10-CH3-13C2] metho-trexate and 15N-labelled [1,3,2-NH2-15N3]folate and [1,3,2-NH2-15N3] methotrexate have been synthesized. The two 15N-labelled compounds have been used to directly measure the pKa of N-1, N-3 and 2-NH2 for folate and methotrexate, respectively. It was found that the pKa''s are 5.46, 5.65, and 5.25 for N-1, N-3 and 2-NH2 of methotrexate, respectively, and are 8.17, 8.20 for N-1 and N-3 of folate, respectively. The chemical shifts of 2-NH2 of folate within the pH range of 6.14 and 10.62 show almost no change, indicating no protonation takes place within this pH range. 13C-NMR was employed to monitor the pH-induced conformational change of dihydrofolate reductase (DHFR)/[2,N10-CH3-13C2] methotrexate complex. In all pHs, only one 13C signal was observed for C-2, while two signals resonating at 40.97 and 40.70 ppm (designated as form II and form I, respectively) were observed for N10-CH3 carbon at pH below 7.5, revealing MTX binds in two conformation forms and the protein interactions with C-2 region of pteridine ring are very similar in the two forms. The relative population of these two conformers varies with pH in a complex behavior. The intensity ratio of I40.97/I40.70 increases with the increasing pH. Interestingly, at pH higher than 7.5 a third signal resonating at 40.75 ppm was observed. This signal is different from that of N10-CH3 of free MTX, indicating the presence of a third conformer designated form Ia. We found that the population of this conformer increases as pH increases. The population of form II was independent of pH, when pH is higher than 7.5. These results indicate a pH-induced slow conformational change at higher pH for conformer I. Based on our observation, the complex behavior of DHFR/MTX complex is very likely as this : MTX interacts with DHFR in a non-productive conformation with N1-C8a-C9 as a pivot and the rest of molecules exerting a slow vibrational motion, and the conformational change for pteridine ring and p-aminobenzoyl ring is independent. On the other hand, only one set of 13C-signal was observed when NADP+ was added to the DHFR/MTX binary complex, indicating only a single conformation exists in ternary complex. For the mutant enzyme, D27N, only one conformer was observed for both DHFR/MTX and DHFR/MTX/NADP+ complexes, indicating Asp-27 plays a critical role in DHFR-MTX interaction. The isotope exchange reactions of malonic acid and a malonate ion were investigated by 1H-NMR spectroscopy in acidic and basic D2O solutions, respectively,. The isotope exchange reaction of malonic acid was inhibited by the presence of DNO3 (0-3 M) and DSO4- ion (0-0.1 M), whereas it was catalyzed by the presence of DSO4- ion (30.2 M), D3PO4, D2PO4- ion, DPO4-2 ion. The order of relative reactivity for catalyzing the isotope exchange reaction of malonic acid in D2O is DPO4-2 > D2PO4- > D3PO4 > DSO4- >> DNO3. The rate of the isotope exchange reaction of malonate ion in D2O decreased to a minimum and then increased with increased [NaOD]o. The mechanism of the isotope exchange reaction of malonic acid in acidic D2O is different from the general acid-catalyzed mechanism generally observed for organic acids such as acetic acid and dichloroacetic acid. The kinetics of the isotope exchange reactions of RCH(COOH)2 (R = H, D, Br, Ph, Me, Et, Bu) in D2O solution were also studied by 1H-NMR spectroscopy. It was observed that the rate of the isotope exchange reaction was inhibited by the presence of 1 M DNO3, DCl, DBr, or D2SO4 and catalyzed by the presence of 4 M D2SO4. No inhibition effect was observed in the presence of D3PO4. The effect of inorganic acids follows the order of D3PO4 > D2SO4 >> (DNO3, DCl, DBr). The conjugated base (RCH(COOD)(COO-)) of RCH(COOH)2 plays an important role in the isotope exchange reaction. The presence of deuterium ion suppresses the generation of RCH(COOD)(COO-) ion from RCH(COOH)2 and inhibits the rate of the isotope exchange. In general, the order of reactivity of RCH(COOH)2 toward isotope exchange with deuterium atom is R = Ph > (H, Br) > Me > (Et, Bu). The isotope exchange reaction of ethyl hydrogen malonate (C2H5COOCH2COOK) was catalyzed by the presence of DNO3, DSO4- ion, D3PO4, D2PO4- ion, DPO4-2 ion. The order of relative reactivity for catalyzing the isotope exchange reaction of ethyl hydrogen malonate in D2O is DPO42- > D2PO4- > (DSO4-，D3PO4) > DNO3. The mechanism of the isotope exchange reaction of ethyl hydrogen malonate in acidic D2O is similar to the general acid-catalyzed mechanism generally observed for organic acids. In basic D2O solution, it is observed that ethyl hydrogen malonate undergoes both the isotope exchange reaction and the hydrolysis reaction. The rate of the isotope exchange reaction is considerably higher than that of the hydrolysis reaction. The rate of the hydrolysis reaction of C2H5COOCH2COOK in D2O increases linearly with increasing [NaOD]o (0.1-0.25 M).