Elsevier

Methods in Enzymology

Volume 401, 2005, Pages 449-467
Methods in Enzymology

Gamma‐Glutamyl Transpeptidase Substrate Specificity and Catalytic Mechanism

https://doi.org/10.1016/S0076-6879(05)01027-XGet rights and content

Abstract

The enzyme γ‐glutamyltranspeptidase (GGT) is critical to cellular detoxification and leukotriene biosynthesis processes, as well as amino acid transport in kidneys. GGT has also been implicated in many important physiological disorders, including Parkinson's disease and inhibition of apoptosis. It binds glutathione as donor substrate and initially forms a γ‐glutamyl enzyme that can then react with a water molecule or an acceptor substrate (usually an amino acid or a dipeptide) to form glutamate or a product containing a new γ‐glutamyl isopeptide bond, respectively, thus regenerating the free enzyme. Given the importance of GGT in human physiology, we have undertaken studies of its substrate specificity and catalytic mechanism. In the course of these studies, we have developed methods for the indirect evaluation of donor substrate affinity and stereospecificity and applied others for the measurement of steady state and pre‐steady state kinetics and linear free‐energy relationships. These methods and the pertinent results obtained with them are presented herein.

Introduction

γ‐Glutamyl transpeptidase (GGT; EC 2.3.2.2) is a highly glycosylated heterodimeric enzyme found in mammals and plants (Kasai 1980, Taniguchi 1998). In mammals, it is found in brain, liver, pancreas, and especially in kidneys (Hanigan 1985, Sian 1994, Tate 1985). It plays a role in cellular detoxification through formation of mercapturic acids and confers resistance against anti‐tumor drugs (Godwin et al., 1992). GGT is involved in the biosynthesis of leukotriene D, which mediates bronchoconstriction in asthma (Bernström 1982, Örning 1980) and in amino acid transport in kidneys (Meister, 1973). It has also been implicated in many physiological disorders, such as Parkinson's disease (Sian et al., 1994), diabetes (Lee et al., 2003), and inhibition of apoptosis (Del Bello 1999, Graber 1995). GGT uses glutathione (GSH) as an acyl donor substrate and transfers its γ‐glutamyl moiety to acceptor substrates, such as amino acids or dipeptides, to form a product containing a new isopeptide bond. This transamidation role is very important for amino acid transport in the kidney (Griffith 1978, Meister 1973). The reaction catalyzed by GGT is known to proceed through a modified ping‐pong mechanism, as shown in Scheme 1 (Allison 1985, Taniguchi 1998). The enzyme binds its donor substrate, is transiently acylated by its γ‐glutamyl moiety, and releases the first reaction product (cysteinylglycine, in the case of GSH) during the acylation step. The resulting γ‐glutamyl acyl‐enzyme can then react with either water (hydrolysis) or an acceptor substrate (typically an amino acid or a dipeptide) in a deacylation step to form either glutamate or a transpeptidated product, respectively.

Section snippets

Donor and Acceptor Substrate Affinities and Potential Amino Acid Interactions

Rat kidney GGT catalyzes the cleavage of the γ‐glutamyl bond of GSH and other γ‐glutamyl amides. The γ‐glutamyl moiety of donor substrates is critical for recognition by GGT, whereas the primary amine‐leaving group liberated as the first product in the ping‐pong mechanism can vary from Cys‐Gly, in the case of GSH, to p‐substituted anilines (Ménard et al., 2001). The stereoselectivity for donor substrates having the l‐configuration at their α‐carbon is less elevated than that shown for acceptor

Acyl‐Enzyme Intermediate

As mentioned in the Introduction, GGT catalysis is thought to proceed through a modified ping‐pong mechanism (Scheme 1), as evidenced by parallel Lineweaver‐Burk plots (Elce 1976, Tate 1974). However, it has been suggested that inhibition caused by the binding of acceptor substrates into donor substrate binding sites at high concentrations may also provide parallel plots for a sequential mechanism (Allison 1985, Gololobov 1994). The isolation of the acyl‐enzyme intermediate typical for

Binding Site Mapping through Competition Experiments

Using 0.1 M MOPS pH 7.0 buffer as a solvent, stock solutions of 25–500 mM in amide donor substrate analogues were prepared. Subsequent kinetic studies were carried out using different concentrations of l‐γ‐glutamyl‐p‐nitroanilide (between 50 and 1680 μM), 20 mM glycylglycine, and different concentrations of amide substrate (usually from 0 up to 100 mM when possible) in 0.1 M MOPS pH 7.0 buffer at a final volume of 1 mL. Reactions were initiated by adding 3.38 mU of GGT. The liberation of p

Acknowledgments

We thank the Natural Sciences and Engineering Research Council (NSERC) of Canada for financial support. We thank also the NSERC for a postgraduate scholarship (RC) and the Université de Montréal for a Bourse d'excellence (CL).

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    These authors contributed equally to this work.

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