Abstract
The enzyme gamma-glutamyl transpeptidase (GGT) has been investigated in detail, with respect to both its catalytic action and its involvement in amino acid translocation across cell membranes. A novel source of this enzyme was discovered, Caco-2 cell monolayers which show similar characteristics to those of the small intestine. The catalytic action of the enzyme in the cells, was shown to be similar to that of an isolated purified GGT, (bovine kidney) obtained commerical1y. The Caco-2 cell monolayers were used as an in vitro model to study the translocation of amino acids and dipeptides across these biological membranes. The movement of selected substrates was studied using radiochemical techniques. These studies strongly suggest the existence of two distinct transport pathways: an active pathway which is unidirectional, carrier-mediated, saturable, temperature dependent and some way dependent on GGT; a second translocation pathway which is passive, bidirectional, temperature independent and not directly affected by GGT. This evidence is the strongest thus far, to suggest the involvement of GGT, in the translocation of amino acids. However, it cannot be established if GGT is directly involved, ie: via the gamma-glutamyl cycle or whether indirectly via the catalytic formation of a 'messenger' which regulates the passive pathway. A series of gamma-glutamyl donors, acceptors and inhibitors, were synthesised and used to probe the active site of the catalytic action of this enzyme. Studies of the two halves of the ping-pong mechanism via a gamma-glutamyl-enzyme intermediate allowed probing of the gamma-glutamyl donor and the acceptor site respectively. Evidence for three distinct binding sites was shown, which could be fitted to the shape of glutathione, probably the natural donor. The gamma-glutamyl donor was shown to be non-stereospecifc, although its specificity towards chain length was shown to be greater than 104, with respect to ?-aspartyl and gamma-glutamyl. The site was shown for the first time to require both an a-amino and an a-carboxylic acid functional group to undergo binding. The only type of gamma-glutamyl donors were those with a secondary gamma-amide bond. In contrast the acceptor site was shown to be stereospecific, only allowing L-isomers to undergo transpeptidation, while only requiring an a-amino and an a-carbonyl group. The acceptor capability of a particular substrate, is dependent upon two factors. Firstly the pK of its a-amino group, since the pH of the solution will determine the amount of free base present, which is able to attack the gamma-glutamyl-enzyme intermediate. The second factor is governed by its structure, if it binds only to the acceptor or whether it binds to both the acceptor and gamma-glutamyl binding sites, thus inhibiting. It was also demonstrated that substrates which are able to interact with the carboxy terminus binding site, ie: the third site, have a cooperative effect on the gamma-glutamyl binding site, thus stimulating the hydrolysis of donors. Such compounds include maleic acid, N-acetyl-glycine, aminoacyl-glycines and certain donors ie: glutathione and gamma-glutamyl-p-nitroani1ide.