Kinetic Characterization and Identification of the Acylation and Glycosylation Sites of Recombinant Human -Glutamyltranspeptidase

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• 作者：Roselyne Castonguay ; Dany Halim ; Myl&egrave ; ne Morin ; Alexandra Furtos ; Christian Lherbet ; Eric Bonneil ; Pierre Thibault ; Jeffrey W. Keillor
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：October 30, 2007
• 年：2007
• 卷：46
• 期：43
• 页码：12253 - 12262
• 全文大小：274K
• 年卷期：v.46,no.43(October 30, 2007)
• ISSN：1520-4995

文摘

-Glutamyltranspeptidase (GGT) is a heterodimeric enzyme important for glutathionehomeostasis control. It has also been implicated in many physiological disorders, including Parkinson'sdisease, apoptosis inhibition, and diabetes. In the first step of its ping-pong mechanism it binds glutathione,its in vivo substrate, and releases cysteinylglycine upon formation of an acyl-enzyme intermediate. Thisintermediate can then react with water to release glutamate as a hydrolysis product or with an amino acidor dipeptide to form a transpeptidation product. Further detailed study of the mechanism underlying thesereactions is hindered at least for some GGTs by the low quantities of protein available after a multisteppurification from tissue. In the present work the gene for human GGT was cloned into the pPICZAvector and transformed into Pichia pastoris to express as a 68 kDa His-tagged protein. The optimizedexpression and secretion of this enzyme in 1 L of culture and subsequent purification by immobilizedmetal affinity chromatography yielded 1.6 mg of purified enzyme having a specific activity of 237 U/mg.Kinetic parameters for the transpeptidation reaction between glutathione and glycylglycine were determinedby mass spectrometry, giving a k_cat of 13.4 × 10³ min^-1 and apparent K_M values of 1.11 mM for glutathioneand 8.1 mM for glycylglycine. The GGT-mediated hydrolysis of glutathione was also studied, providinga k_cat of 53 min^-1 and a K_M value of 7.3 M for glutathione. Incubation of the enzyme with a mechanism-based inhibitor, enzymatic digest, and mass spectrometric analysis provided the first unambiguousidentification of Thr381 as the active site nucleophile of human -glutamyltranspeptidase, and confirmedfour of the seven N-linked glycosylation sites. These structural and kinetic data are discussed with respectto a homology model generated to facilitate visualization.