gamma-Glutamyl transpeptidase acylation with peptidic substrates: free energy relationships measured by an HPLC kinetic assay.
gamma-Glutamyl transpeptidase (GGT, EC 2.3.2.2) is a highly glycosylated heterodimeric enzyme linked to the external cellular membrane that catalyzes the hydrolysis of glutathione as well as the transfer of its gamma-glutamyl group to amino acids and dipeptides in a transpeptidation reaction. The measurement of both the hydrolytic and transpeptidation activity of this important enzyme is a challenge, since its native substrates are not highly chromogenic. We have developed an HPLC-based method for the quantitative photometric detection of numerous enzyme substrates and products, after their pre-column derivation with dabsyl chloride. The broad applicability of this method was demonstrated in the kinetic investigation of transpeptidation reactions of rat kidney GGT with glutathione, its native substrate, as well as a series of pertinent glutathione analogues. The pH-rate profile constructed for glutathione confirmed the dependence on the ionisation state of at least two residues. Analysis of the free-energy relationships in the series of synthetic peptidic substrate analogues revealed the importance of enzyme-substrate interactions unrelated to amine leaving group basicity during the acylation step. These results are further interpreted in the context of the recently published structure for a similar GGT.
Morin
Mylène
M
Département de Chimie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, QC H3C 3J7, Canada.
Rivard
Caroline
C
Keillor
Jeffrey W
JW
D000215:Acylation; D000577:Amides / Q000138:chemical synthesis; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D018360:Crystallography, X-Ray; D005978:Glutathione / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013379:Substrate Specificity; D013816:Thermodynamics; D005723:gamma-Glutamyltransferase / Q000378:metabolism*
D000577:Amides; D010455:Peptides; D005723:gamma-Glutamyltransferase; D005978:Glutathione