Structural study of active center of γ-glutamyltranspeptidase and its reaction mechanism.

γ-谷氨酰转肽酶（GGT）利用D-和L-γ-谷氨酰化合物作为底物。用D/L-谷氨酰胺和D/L-γ-谷氨酰-对硝基苯胺测定动力学参数。对D类化合物的Km值约为对L类化合物的10倍。这表明在α碳上存在同时识别氨基和羰基的残基。GGT与IV类头孢菌素酰化酶的氨基酸序列具有很高的相似性。特别是，GGT中保守的残基在IV类头孢菌素酰化酶中非常保守。其中，Asp-433的非GGT残基可能与γ-谷氨酰化合物的α-氨基相互作用，由于该残基在IV类头孢菌素酰化酶中为Asn，故将其用Asn取代。突变体GGT获得GL-7-ACA酰化酶活性，我们成功地将GGT转化为IV类头孢菌素酰化酶。将突变体GGT中的相应残基用Ala取代后，突变体GGT失去转肽活性，但仍具有水解活性。使用GGT晶体，我们已经模拟了90%的大肠杆菌GGT残基。

γ-Glutamyltranspeptidase(GGT) utilizes both D and L-γ-glutamyl compounds as substrates. Kinetic parameters were measured using D/L-glutamine and D/L-γ-glutamyl-p-nitroanilide. Km values against D compounds were about 10 times bigger that that against L-compounds. This indicates that there are residues recognizing both amino group and carbonyl group on α carbon. The amino acid sequences of GGTs and class IV cephalosporin acylases have high similarity. Especially, residues conserved among GGTs are well conserved in class IV cephalosporin acylases. Among them, Asp-433, whose corresponding residue inhuman GGT was suggested to interact with α-amino group of γ-glutamyl compounds, was substituted with Asn since this residue is Asn in class IV cephalosporin acylases. The mutant GGT obtained GL-7-ACA acylase activity and we succeeded in converting GGT to class IV cephalosporin acylase. When the corresponding residue in Bacillus subtilis GGT was substituted with Ala, the mutant GGT lost the transpeptidation activity while hydrolysis activity was remained. Using GGT crystals, we had modeled 90% residues of E.coli GGT.