Gamma Glutamyl Transpeptidase from Brevibacillus Brevis: Production, Biochemical Characterization and Expression in E. Coli

Chapter 1

INTRODUCTION

γ- Glutamyl transpeptidase (GGT) [E.C. 2.3.2.2], a heterodimeric enzyme, consisting of large and small subunits is involved in the metabolism of glutathione (Taniguchi & Ikeda, 1998). It catalyses the cleavage of γ-glutamyl linkage present in glutathione (GSH), GSH S-conjugates, and other γ-glutamyl compounds and transfers their γ-glutamyl moiety to acceptors such as amino acids, dipeptides, and H20. GGT plays a key role in the gamma-glutamyl cycle, a pathway for the synthesis and degradation of glutathione (Courtay C et al, 1992).

GGT is evolutionary conserved and found in organisms ranging from bacteria to mammals. Prokaryotic and eukaryotic GGTs differ in both structural and functional aspects, however, in both, the two polypeptide chains are processed from a single chain precursor by an autocatalytic cleavage. The active site of GGT is known to be located in the smaller subunit. While prokaryotic GGTs occur as soluble proteins either in the periplasmic (Suzuki et al , 2002) or extracellular space (Minami et al, 2003), the eukaryotic homologues are type II trans-membrane proteins. In mammals, it is found in many tissues, the most notable one being in the liver. It has significance in medicines as a diagnostic marker because its activity is elevated in many diseases including liver diseases, cardiovascular disease, diabetes mellitus, and cancers. In mammals, GGT is involved in the conversion of leukotriene C4 to D4, although it is not the only enzyme that catalyses this reaction (Brucke et al, 2000). Genetic diseases of GGT deficiency, including glutathionuria and glutathionemia, are associated with mental retardation. Clinically, GGT is used in a blood test and, because GGT in serum is mainly derived from liver, high levels of GGT in the blood is indicative of hepatic or biliary tract-associated diseases. Additionally, GGT is a virulence factor associated with colonization of Helicobacter pylori. Thus, it is a universal enzyme playing pivotal role in physiology amongst all living systems.

GGT appears to be involved both positively and negatively in the physiology of oxidative stress. The enzyme protects the cells against oxidative effects by its involvement in glutathione synthesis. Induction of oxidative stress results in up regulated expression of GGT. Many substances, including drugs, carcinogens, and alcohol, are also capable of increasing GGT gene expression in various cells and tissues (Brown et al, 1998). Some amino acids and peptides, which have low solubility in water, become much more soluble following γ-glutamylation and more stable in the blood stream. Since the γ-glutamyl linkage cannot be cleaved by normal peptidases in serum, the half lives of compounds become much longer with γ-glutamylation.

Some of the common substrates for invitro detection of GGT are glutathione, glutamine, and γ-glutamyl-p-nitro aniline. Of these, the latter is the most popular substrate for GGT activity analysis. Reaction proceeds with the hydrolysis of the molecule into glutamic acid and p-nitro aniline. The bright yellow colour of 4-nitroaniline (ε410=8800M-1) allows quantification by spectrophotometry. Glycyl-glycine is used as the standard acceptor for analysing the kinetics of trans-peptidation.

REACTION MECHANISM

The enzymatic reaction catalysed by GGT proceeds via a glutamyl enzyme intermediate. The activated oxygen atom of the side chain of the N-terminal threonine residue of the small subunit of GGT attacks the carbonyl carbon of a γ-glutamyl compound to form a γ-glutamyl enzyme intermediate. When this intermediate is subjected to nucleophilic substitution by amino acids or peptides, the reaction is a transpeptidation reaction, producing new γ-glutamyl compounds. When the intermediate is subjected to nucleophilic attack by water, the reaction is a hydrolysis reaction, releasing glutamate (Suzuki et al, 2013).

Three types of reactions are possible based on the destination of the γ-glutamyl moiety:

1. Transfer to water results in hydrolysis

2. Trans-peptidation reaction ensues on transfer to an “acceptor” like amino acids or peptides.

3. Transfer to another molecule of the substrate results in auto-transpeptidation.

ROLE OF γ- GLUTAMYL TRANSPEPTIDASE IN γ-GLUTAMYL CYCLE

γ-glutamyl transpeptidase has been proposed to play a physiological role in the γ-glutamyl cycle which is involved in the uptake of amino acids through the cell membrane into mammalian and bacterial cells (Orlowski and Meister, 1970).

The γ-glutamyl cycle, a biochemical mechanism proposed by Meister, describes the metabolism of glutathione. The first step in the metabolism of glutathione occurs in the extracellular environment and involves the enzyme, γ –glutamyl transpeptidase (Meister and Tate. 1976). γ –glutamyl transpeptidase is the only known enzyme that has the capability to cleave intact glutathione (Meister and Tate, 1976; Meister et al., 1981; Hanigan, 1998). When glutathione encounters γ –glutamyl transpeptidase, the γ-glutamyl bond of the glutathione molecule is hydrolysed by γ–glutamyl transpeptidase .The products that form as a result of this interaction are γ-glutamyl moiety and a cysteinyl-glycine molecule. The following reactions can occur through the breakdown of glutathione:

1. γ-glutamyl-cysteinyl-glycine + amino acid γ-glutamyl-amino acid + cysteinyl-glycine

2. γ-glutamyl-cysteine-glycine + water glutamate + cysteine-glycine

3. γ-glutamyl-cysteine-glycine + γ-glutamyl-cysteine-glycine γ-(γ-glutamyl)-glutamyl-çysteine-glycine + cysteine-glycine

If there is an abundance of an acceptor molecule, such as an amino acid or a dipeptide, the γ-glutamyl moiety can bind to the acceptor and be transported into the ceIl (Reaction 1). This is known as transpeptidation (Allison et al, 1985).

Water can serve as an acceptor for the γ-glutamyl moiety resulting in hydrolysis of the γ -glutamyl bond (Reaction 2) to produce glutamate and cysteine-glycine.

In the absence of an acceptor (as seen in Reaction l) or water (as seen in Reaction 2), auto-transpeptidation can occur (Reaction 3) where the γ- glutamyl moiety can bind to another glutathione molecule leaving cysteine-glycine as the other product (Abbott et al, 1986). The cysteine-glycine product can then be cleaved by peptidases, such as amino-peptidases, found in the extracellular

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