Slow-binding inhibition of gamma-glutamyl transpeptidase by gamma-boroGlu.
gamma-Glutamyl transpeptidase (gammaGTase) catalyzes the transfer of the gamma-glutamyl moiety of gamma-glutamyl-derived peptides, such as glutathione (gammaGlu-Cys-Gly), and anilides, such as gamma-glutamyl-7-amido-4-methylcoumarin (gammaGlu-AMC), to acceptor molecules, including water and various dipeptides. These acyl-transfer reactions all occur through a common acyl-enzyme intermediate formed from attack of an active site hydroxyl on the gamma-carbonyl carbon of gammaGlu-X with displacement of X. In this paper, we report that gammaGTase is potently inhibited by the gamma-boronic acid analogue of L-glutamic acid, 3-amino-3-carboxypropaneboronic acid (gamma-boroGlu). We propose that gamma-boroGlu adds to the active site hydroxyl of gammaGTase to form a covalent, tetrahedral adduct that resembles tetrahedral transition states and intermediates that occur along the reaction pathway for gammaGTase-catalyzed reactions. Our studies demonstrate that gamma-boroGlu is a competitive inhibitor of the gammaGTase-catalyzed hydrolysis of gammaGlu-AMC with a K(i) value of 35 nM. Kinetics of inhibition studies allow us to estimate the following values: k(on) = 400 mM(-1) s(-1) and k(off) = 0.02 s(-1). We also found that gamma-boroGlu is an uncompetitive inhibitor of Gly-Gly-promoted transamidation of gammaGlu-AMC. This observation is consistent with the kinetic mechanism we determined for gammaGTase-catalyzed transamidation of gammaGlu-AMC by Gly-Gly to form gammaGlu-Gly-Gly. To probe rate-limiting transition states for gammaGTase catalysis and inhibition, we determined solvent deuterium isotope effects. Solvent isotope effects on k(c)/K(m) for hydrolysis of gammaGlu-AMC and k(on) for inhibition by gamma-boroGlu are identical and equal unity, suggesting that the processes governed by these rate constants are both rate-limited by a step that is insensitive to solvent deuterium such as a conformational fluctuation of the initially formed E-S or E-I complex. In contrast, the solvent isotope effect on k(c) is 2.4. k(c) is rate-limited by hydrolysis of the acyl-enzyme intermediate that is formed during reaction of gammaGTase with gammaGlu-AMC. Thus, the magnitude of this isotope effect suggests the formation of a catalytically important protonic bridge in the rate-limiting transition state for deacylation.
Stein
R L
RL
Department of Chemical Enzymology, DuPont Pharmaceuticals Company, Wilmington, Delaware 19880, USA. Ross.L.Stein@dupontpharma.com
DeCicco
C
C
Nelson
D
D
Thomas
B
B
D000215:Acylation; D000577:Amides / Q000378:metabolism; D001667:Binding, Competitive; D001896:Boron Compounds / Q000378:metabolism*; D001897:Boronic Acids / Q000378:metabolism; D002384:Catalysis; D003374:Coumarins / Q000378:metabolism; D003903:Deuterium; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000031:analogs & derivatives* / Q000378:metabolism*; D005998:Glycine / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D012997:Solvents; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism*
C426904:2-amino-4-boronobutanoic acid; D000577:Amides; D001896:Boron Compounds; D001897:Boronic Acids; D003374:Coumarins; D012997:Solvents; D018698:Glutamic Acid; C027245:7-(gamma-glutamyl)-4-methylcoumarylamide; D003903:Deuterium; D005723:gamma-Glutamyltransferase; D005998:Glycine