Kinetic studies of rat kidney gamma-glutamyltranspeptidase deacylation reveal a general base-catalyzed mechanism.
The enzyme gamma-glutamyltranspeptidase (GGT) is critical to cellular detoxification and leukotriene biosynthesis processes, as well as amino acid transport in kidneys. GGT has also been implicated in many important physiological disorders, including Parkinson's disease and inhibition of apoptosis. It binds glutathione as a donor substrate and initially forms a gamma-glutamyl-enzyme complex that can then react with a water molecule or an acceptor substrate (usually an amino acid or a dipeptide) to form glutamate or a product containing a new gamma-glutamyl-isopeptide bond, respectively, thus regenerating the free enzyme. Despite its important role in human physiology, the mechanisms of the reactions catalyzed by GGT are not well-known, particularly with respect to the deacylation step. We have synthesized a series of methionine amide derivatives whose alpha-ammonium groups have different pK(a) values. By using these compounds as acceptor substrates for GGT, we have constructed a Brønsted plot and obtained a good correlation for log(k(norm)(cat,b)/K(b)) versus pK(a)(NH+) with a slope beta(nuc) of 0.84, consistent with a rate-limiting nucleophilic attack of the substrate amine on the acyl-enzyme intermediate. Isotope effect studies have shown that there is a proton in flight at the transition state, consistent with concerted deprotonation of the nucleophilic amine effected by an unidentified general base. A bell-shaped pH-rate profile has also been obtained for the deacylation step, reflecting the pK(a) values of the acceptor substrate (and/or that of a general base residue) and of a putative general acid that may be necessary for reprotonation of the active site nucleophile upon regeneration of the free enzyme. These data allow us to propose for the first time a detailed mechanism for this important step of the GGT-mediated reaction and to speculate about the origin of its acceptor substrate specificity.
D000215:Acylation; D000577:Amides / Q000737:chemistry / Q000378:metabolism; D000588:Amines / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008715:Methionine / Q000031:analogs & derivatives / Q000378:metabolism; D011522:Protons; D000644:Quaternary Ammonium Compounds / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D005723:gamma-Glutamyltransferase / Q000737:chemistry* / Q000378:metabolism*
D000577:Amides; D000588:Amines; D011522:Protons; D000644:Quaternary Ammonium Compounds; D008715:Methionine; D003903:Deuterium; D005723:gamma-Glutamyltransferase