γ-GT

Gamma-glutamyl transferase (γ-GT, GGT, EC 2.3.2.2) is a transferase that catalyzes the transfer of gamma-glutamyl groups from molecules such as glutathione to an acceptor such as an amino acid, a peptide or water. γ-GT plays a key role in the gamma-glutamyl cycle by regulating the cellular levels of the antioxidant molecule glutathione, hence it is a critical enzyme in maintaining cellular redox homeostasis. γ-GT has been widely used as an index of liver dysfunction and marker of alcohol intake. There have also been important advances in the definition of the associations between serum GGT and risk of coronary heart disease, Type 2 diabetes, and stroke.

Structure

The primary structures of γ-GTs from various sources have been identified, ranging from bacteria to mammals. Sequence alignments point to a strong conservation of structure and function in these enzymes, which share >25% identity. γ-GT polypeptide chain undergoes an autoproteolytic cleavage into a large and a small subunit. The molecular weights of the two chains are generally found to be within 38–72 kDa for the large subunit and within 20–66 kDa for the small subunit. In mammalian, the large subunit has an intracellular N-terminal sequence, a single transmembrane domain anchored to membrane by a hydrophobic fragment of eight residues and a large extracellular domain that binds the small subunit. The extracellular component can be separated from the membrane by papain treatment. The hydrophobic anchor can be removed without affecting the enzyme activity.

Figure 1. Structure of E. coli GGT. (Okada T. et al. 2006)

X-ray crystallography has played a crucial role in the study of γ-GTs, providing researchers with detailed structural models that have been essential to unveil the enzyme function and reactivity. All γ-GTs belong to the superfamily of the N-terminal nucleophilic hydrolase which exhibit a similar structure. The members of this superfamily share a common active site architecture, tertiary and quaternary fold, although they can show pairwise sequence identities as low as 15%. The predominant elements of secondary structure in nucleophilic hydrolases are a long four-layer αββα-structure, with two antiparallel β-sheets packed against each other and sandwiched between α-helical layers. In the central β-sheet sandwich, both large and small subunits provide strands. They are arranged in an antiparallel fashion: one of the two central sheets is essentially flat, whereas the other may be twisted at the end. In the studied γ-GTs, the small and the large subunits are highly intertwined throughout the structure. The inter-subunit interface is stabilized by both hydrogen bonds and hydrophobic contacts.

Function

Evidence of the normal function of γ-GT has come from multiple sources, some circumstantial and some direct. These include the location of the enzyme at membrane transport sites, where it has been detected by histochemical or immunohistochemical methods; experiments using γ-GT inhibitors such as acivicin and serine-borate, cell transfection, knockout animals and human subjects with γ-GT deficiency; and study of the response of γ-GT enzyme, protein, or mRNA concentrations to the manipulation of glutathione status.

The most abundant substrate for γ-GT is glutathione, and its physiological roles relating to glutathione have been investigated extensively. One of the early observations about γ-GT was that its activity was greatest in tissues with a transport function, such as the kidneys and in the biliary system. This led to the suggestion that γ-GT played an important role in the transport of amino acids, through a sequence of reactions forming a “gamma-glutamyl cycle”. The γ-glutamyl cycle utilizes glutamate (GSH) as a continuous source of cysteine for the cell. It is synthesized in the cytosol and is then translocated out of the cell. Extracellular GSH metabolism is initiated by the γ-GT, the first enzyme of the GSH degradation pathway, and then completed by membrane dipeptidases.

Figure 2. Gamma-glutamyl cycle. (Castellano I. et al. 2013)

Activity Measurement

The reaction catalyzed by γ-GT is the transfer of a glutamyl residue, linked through glutamate’s gamma carboxylic acid to an amine or to another amino acid, to an acceptor. The γ-GT reaction takes the general form: Gamma-glutamyl-X + acceptor → Gamma-glutamyl-acceptor + X, and a wide range of compounds can participate as the gamma-glutamyl donor or as the acceptor. Among the gamma-glutamyl donors the most significant natural substrate is believed to be glutathione, but a number of artificial substrates have been developed for convenient measurement of γ-GT activity. These include gamma-glutamyl-β-naphthylamide, gamma-glutamyl-p-nitroanilide, and gamma-glutamyl-3-carboxy-4-nitroanilide. The nitroanilide have the advantage of being chromogenic substrates, and the progress of the reaction can be monitored continuously. The carboxylic acid avoids some solubility limitations that occur with gamma-glutamyl-p-nitroanilide. γ-GT activity is usually measured by kinetic spectrophotometric methods based on the work of Szasz, which are precise and inexpensive.

Immunoassays, and high-sensitivity methods based on fluorescence, have also been described for specialized uses. The study of isoenzymes has led to the conclusion that changes in the carbohydrate composition of γ-GT affect association with lipoproteins and produce variation in electrophoretic patterns, but isoenzyme analysis has not been widely used because of technical inconvenience and lack of evidence for diagnostic usefulness.

Liver Diseases

γ-GT was investigated and adopted as a liver function test or liver enzyme. An early paper by Szczeklik et al. gave average values in different types of liver disease and showed examples of changes in serum γ-GT with time, comparing it with other enzymes. It is a sensitive test, being abnormal in most patients with liver disease regardless of cause, although higher values are found in patients with cholestasis. The problem lies in a lack of specificity, given the wide range of diseases or other conditions (such as pancreatitis, diabetes, obesity, excessive alcohol intake, and use of enzyme-inducing drugs) that can also cause high serum γ-GT.

Apart from its general use in the assessment of liver disease, GGT has been evaluated in a number of common or significant clinical settings. In Porphyria Cutanea Tarda (PCT) there is a very high prevalence of abnormal GGT. GGT decreases with treatment of PCT, whether the treatment is through abstinence from alcohol or iron removal, chloroquine, cimetidine, or following treatment of hepatitis C, which is a known risk factor for PCT. Some porphyrinogenic drugs have the effect of increasing hepatic or serum GGT. Nonalcoholic steatohepatitis (NASH) is a hepatic disease associated with diabetes and obesity and shares many features with alcoholic liver disease. GGT is increased in most patients with this condition, but it is not of diagnostic use. The main interest in relation to GGT is the fact that the condition is similar to alcoholic liver disease but without the alcohol, and that it may represent the extreme end of the epidemiological associations between GGT and obesity, insulin resistance, diabetes, and fatty liver.

References