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.
Highlights
Glutathione S-transferases (GSTs, EC 2.5.1.18) are a superfamily of multifunctional enzymes involved in cellular detoxification by conjugating the tripeptide glutathione (GSH) to a wide range of endobiotic and xenobiotic electrophilic compounds [12,13,14]
ScFv2F3 displays glutathione S-transferases (GSTs) activity, indicating that in scFv2F3 there is an active site that could bind the substrates GSH and CDNB and has a catalytic group to catalyze the conjugation of GSH to CDNB
Which amino acid residue is the catalytic residue of scFv2F3? The experimental result showed that when scFv2F3 was converted into Se-scFv2F3 by chemical mutation of hydroxyl groups in the Ser residues of scFv2F3, it lost all GST activities and gained a high glutathione peroxidase (GPX) activity to CuOOH or H2O2 [6], indicating that Ser in scFv2F3 could greatly contribute to GPX activity [4]
Summary
ND 102 a All values are the means of at least five determinations. b ND, no detectable GST or GPX activity. c The GST or GPX activities of rGST T2–2 and Se-rGST T2–2 are from Ref. 4. The activity of GST toward CDNB was measured in 50 mM potassium phosphate buffer (pH 6.5) containing 1 mM EDTA, 1 mM CDNB, and 1 mM GSH. The activity of GST toward 1-bromo-4nitrobenzene was measured in 50 mM potassium phosphate buffer (pH 6.5) containing 1 mM EDTA, 0.1 mM 1-bromo-4-nitrobenzene, and 5 mM GSH. The reactions were carried out in a total volume of 0.7 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM EDTA, 1 mM sodium azide, 1 mM GSH, 1 unit of GSH reductase, and 10 –50 nM protein. One unit of activity is defined as the amount of protein that catalyzes the turnover of 1 mol NADPH/min. Glutathione S-transferase activity with CDNB was measured in the presence of 50 mM potassium phosphate at pH 6.5, 1 mM GSH, 1 mM CDNB in ethanol (% (v/v) final concentration).
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