Single chain antibody displays glutathione S-transferase activity.
Substrate binding and the subsequent reaction are the two principal phenomena that underlie the activity of enzymes, and many enzyme-like catalysts were generated based on the phenomena. The single chain variable region fragment of antibody 2F3 (scFv2F3) was elicited against hapten GSH-S-DN2phBu, a conjugate of glutathione (GSH), butyl alcohol, and 1-chloro-2,4-dinitrobenzene (CDNB); it can therefore bind both GSH and CDNB, the substrates of native glutathione S-transferases (GSTs). It was shown previously that there is a serine residue that is the catalytic group of GST in the CDR regions of scFv2F3 close to the sulfhydryl of GSH. Thus, we anticipated that scFv2F3 will display GST activity. The experimental results showed that scFv2F3 indeed displayed GST activity that is equivalent to the rat-class GST T-2-2 and exhibited pH- and temperature-dependent catalytic activity. Steady-state kinetic studies showed that the Km values for the substrates are close to those of native GSTs, indicating that scFv2F3 has strong affinities for the substrates. Compared with some other GSTs, its kcat value was found to be low, which could be caused by the similarity between the GSH-S-DN2phBu and the reaction product of GSH and CDNB. These results showed that our approach to imitating enzymes is correct, which is that an active site may catalyze a chemical reaction when a catalytic group locates beside a substrate-binding site of a receptor. It is important to consider product inhibition in hapten design in order to obtain a mimic with a high catalytic efficiency.
Zhang
Kun
K
Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 13002, People's Republic of China.
Zang
Tian-zhu
TZ
Yang
Wei
W
Sun
Ye
Y
Mu
Ying
Y
Liu
Jun-qiu
JQ
Shen
Jia-cong
JC
Luo
Gui-min
GM
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D004137:Dinitrochlorobenzene / Q000737:chemistry; D005978:Glutathione / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D013379:Substrate Specificity; D013696:Temperature
D004137:Dinitrochlorobenzene; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D005982:Glutathione Transferase; D005978:Glutathione