Study on protease which processes gamma-glutamyltranspeptidase

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N-terminal signal peptide of GGT consists of 25 amino acid residues, the large subunit of 365 residues and the small subunit of 190 residues are en coded in a single open reading frame in this order and this is a very rare gene construct. This suggests that besides the cleavage by signal peptidase I,E.coli GGT is subjected to a post-translational cleavage between Gln-390 and Thr-391 and is matured into a heterodimer as mammalian GGTs. Sequence alignment for similarity at the processing site among GGTs and related enzymes was performed. Gln-390, the C-terminal amino acid residue of the large subunit, is not conserved, while Thr-391 and His-393, N-terminal amino acid residues of the small subunit, are conserved among all GGTs whose amino acid sequences have been known. Periplasmic fractions of T391A and H393G mutants did not process. Therefore, Thr-X-His sequence of N-terminal of the small subunit is critical for the processing. In mammals there was a hypothesis that GGT is processed by … More a membrane bound trypsin-like serine protease, but no farther report about this protease thereafter. Recently, from the results of three dimensional structure analysis there is a hypothesis that the side chain of N-terminal amino acid residue of the small subunit (Thr-391 in E.coli GGT) is the nucleophilic atom which attacks the carbonyl carbon of peptide linkage between Gln-390 and Thr-391, and the processing takes place. However, no experimental proof has been obtained. From the chemical modification study some basic amino acid residues of mammalian GGTs were suggested to locate in the active center for the enzymatic reaction. Therefore, basic amino acid residues which are highly conserved among GGTs (Arg-513 and Arg-571 in E.coli) were mutagenized by site-directed mutagenesis and their effects were observed. Although these residues are far from the processing site in the primary structure of GGT,these mutants were not subjected to processing and did not have the enzymatic activity. This suggests that the processing occurs after the conformation of GGT has formed. Amino acid residues in C-terminal of B-chain of penicillin acylase which is subjected to the similar post-translational processing are critical. This is one evidence that GGT is processed by the similar way as penicillin acylase which is one of the proposed N-terminal nucleophile hydrolase superfamily. All of members of this family have characteristic two antiparallel beta-pleated sheets consisting of several beta-strands. These enzymes use the side chain of the N-terminal amino acid residue of B-chain as the nucleophile in the catalytic attack at the carbonyl carbon of substrates and this side chain was suggested to be responsible for their autoprocessing mechanism. Although we have not completed X-ray analysis of E.Coli GGT,we have found that GGT also possesses the characteristic two antiparallel beta-pleated sheets. This result strongly suggests that the processing of GGT is autocatalytic and that the Less