|Budget Amount *help
¥3,200,000 (Direct Cost : ¥3,200,000)
Fiscal Year 1999 : ¥1,500,000 (Direct Cost : ¥1,500,000)
Fiscal Year 1998 : ¥1,700,000 (Direct Cost : ¥1,700,000)
In the present study we performed a structural study for two catalytic antibodies, 6D9 and 9C10, which catalyze the hydrolysis of a non-bioactive chloramphenicol monoester derivative (Substrate) to generate chloramphenicol. Although there is a high similarity in primary structures of these antibodies, 6D9 possesses the higher activity (kィイD2catィエD2/kィイD2uncatィエD2 = 895) than 9C10 does (kィイD2catィエD2/kィイD2uncatィエD2 = 56). On the basis of the enzymatic study, the difference in the binding constant of the antibodies for transition state analogue (TSA) and Substrate is responsible for the expression of the catalytic activities. Therefore we decided to investigate how the catalytic antibodies discriminate TSA from Substrate at the atomic level using NMR. From the result of our NMR analyses, we conclude the followings,
1. Imidazole proton of His27d(L) of 6D9 forms a hydrogen bond to the oxygen atom of phosphonate group of TSA, while the hydrogen bond is not found in the 6D9-Substrate, 9CIO-Sub
strate and 9CIO-TSA complexes. His27d(L) of 6D9 plays an important role for discriminating the difference of chemical structure between TSA and Substrate.
2. Unusual broadening observed for aromatic proton resonances of TSA and Substrate in the bound state revealed that a flip-flop motion of 4-((trifluoroacetyl)amino)phenyl ring is restricted in the 9C10-Substrate and 9C10-TSA complexes. The same phenomenon is noted in both of the two aromatic rings of TSA in the 6D9-TSA complex, indicating that a strong interaction exists between the amino acid residues and both of the two aromatic rings in 6D9-TSA complex. Considering that relative positions of the two aromatic rings in the binding site is different between TSA and Substrate since the atomic orbitals of carbonyl carbon in Substrate and phosphorus in TSA are spィイD12ィエD1 and spィイD13ィエD1, respectively, we conclude that 6D9 could recognize the structural difference between TSA and Substrate in the bound state through the interaction and then 6D9 gain the high catalytic activities.
Next, we performed a structural study of catalytic antibody 7C8. In this study we have investigated the microenvironment of the reaction center, carbonyl carbon of Substrate which is bound to 7C8, and determined the pH profile of the 7C8 catalyzed reaction rate. These results indicate that the residue of which pKa is approximately 4.5 affects the catalytic mechanism of 7C8. The pH titration study of 7C8 labeled with ィイD115ィエD1N revealed that the functional residue is His92(L). It is concluded that His92(L) as well as Tyr95(H) play a significant role in catalytic reaction by 7C8. Investigation of catalytic mechanism by these residues is now in progress. Less