title: Single chain antibody displays glutathione S-transferase activity.
abstract: 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.
authors.affiliation: Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, Jilin University, 2519 Jiefang Road, Changchun 13002, People's Republic of China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Tian-zhu
authors.initials: TZ
authors.lastname: Zang
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.forename: Ye
authors.initials: Y
authors.lastname: Sun
authors.forename: Ying
authors.initials: Y
authors.lastname: Mu
authors.forename: Jun-qiu
authors.initials: JQ
authors.lastname: Liu
authors.forename: Jia-cong
authors.initials: JC
authors.lastname: Shen
authors.forename: Gui-min
authors.initials: GM
authors.lastname: Luo
mesh_terms: 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
chemical_list: D004137:Dinitrochlorobenzene; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D005982:Glutathione Transferase; D005978:Glutathione