© 2007 The Japanese Biochemical Society.
Mechanistic Analysis of the Phosphonate Transition-state Analogue-derived Catalytic and Non-catalytic Antibody
1Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512; 2Graduate School of Integrated Science, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama, Kanagawa 236-0027; 3Tobacco Science Research Center, Japan Tobacco, Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512, Japan; and 4Department of Molecular Biology and Biotechnology, Krebs Institute for Biomolecular Research, University of Sheffield, Fifth Court, Western Bank, Sheffield S10 2TN, UK
To whom correspondence should be addressed: Tel: +81-749-64-8122, Fax: +81-749-64-8140, E-mail: y_nishi{at}nagahama-i-bio.ac.jp
Received February 1, 2007; Accepted May 15, 2007
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Abstract |
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The esterolytic catalytic antibody (catAb) has the positive charged region interacting with the carbonyl group of the ester substrate. To examine how such a region interacts with the substrate, we compared the catAb with the non-catalytic antibody (non-catAb) for interaction with the non-cleavable amide substrate (a mimic of the ester substrate) and the two end products. Surface plasmon resonance (SPR) analysis revealed that the amide substrate gave the equivalent Kd values for the two antibodies, whereas both the on-rate and off-rate of the catAb were five-times lower than those of the non-catAb. In agreement with SPR analysis, saturation transfer difference (STD) NMR spectroscopy detected the STD signals only between the catAb and one of the product, suggesting the slower exchange rates of the amide substrate in the catAb as compared with the mixing times, whereas it was not the case with the non-catAb. Transferred nuclear Overhauser effect NMR spectroscopy showed the negative signals for only between the non-catAb and the amide substrate or the product, again suggesting the lower off-rates of the catAb as compared with the mixing times. The decreased interaction rates should be the primary consequence of the positively charged region in the combining site in the catAb.
Key Words: catAb, saturation transfer difference, surface plasmon resonance, transferred nuclear Overhauser effect NMR spectroscopy
Abbreviations: TSA, transition-state analogue; EPS, electrostatic potential surface; SPR, surface plasmon resonance; STD, saturation transfer difference; trNOESY, transferred nuclear Overhauser effect spectroscopy; trNOE, transferred nuclear Overhauser effect; ELISA, enzyme-linked immuno sorbent assay
*Present addresses: Yoshisuke Nishi, Nagahama Institute of Bio-Science and Technology, 1266 Tamura-cho, Nagahama, Shiga 526-0829, Japan; Naoko Takahashi-Ando, Plant Science Center, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Hiroyuki Kakinuma, Medicinary Chemistry Laboratory, Taisho Pharmaceutical Co., Ltd, 1-403 Yoshino-cho, Saitama, Saitama 330-8530, Japan.