Mechanistic analysis of the phosphonate transition-state analogue-derived catalytic and non-catalytic antibody.
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 K(d) 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.
Nishi
Yoshisuke
Y
Laboratory of Life Science & Biomolecular Engineering, Japan Tobacco, Inc., 6-2 Umegaoka, Aoba-ku, Yokohama, Kanagawa 227-8512. y_nishi@nagahama-i-bio.ac.jp
Yamamoto
Naoki
N
Shimazaki
Kazuko
K
Takahashi-Ando
Naoko
N
Kakinuma
Hiroyuki
H
Jialin
Sun
S
Ruzheinikov
Sergey N
SN
Muranova
Tatyana A
TA
Rice
David W
DW
Kajihara
Yasuhiro
Y
D000595:Amino Acid Sequence; D000818:Animals; D017151:Antibodies, Catalytic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001666:Binding Sites, Antibody; D002384:Catalysis; D006241:Haptens / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008807:Mice, Inbred BALB C; D019463:Mice, Inbred MRL lpr; D008969:Molecular Sequence Data; D063065:Organophosphonates / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance
D017151:Antibodies, Catalytic; D006241:Haptens; D063065:Organophosphonates