Slow-binding inhibition of gamma-glutamyl transpeptidase by gamma-boroGlu

Citation
Rl. Stein et al., Slow-binding inhibition of gamma-glutamyl transpeptidase by gamma-boroGlu, BIOCHEM, 40(19), 2001, pp. 5804-5811
Citations number
21
Categorie Soggetti
Biochemistry & Biophysics
Journal title
BIOCHEMISTRY
ISSN journal
00062960 → ACNP
Volume
40
Issue
19
Year of publication
2001
Pages
5804 - 5811
Database
ISI
SICI code
0006-2960(20010515)40:19<5804:SIOGTB>2.0.ZU;2-Q
Abstract
gamma -Glutamyl transpeptidase (gamma GTase) catalyzes the transfer of the gamma -glutamyl moiety of gamma -glutamyl-derived peptides, such as glutath ione (gamma Glu-Cys-Gly), and anilides, such as gamma -glutamyl-7-amido-4-m ethylcoumarin (gamma Glu-AMC), to acceptor molecules, including water and v arious 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 gamma Glu-X with displacement of X. In this p aper, we report that gamma GTase is potently inhibited by the gamma -boroni c acid analogue of L-glutamic acid, 3-amino-3-carboxypropaneboronic acid (g amma -boroGlu). We propose that gamma -boroGlu adds to the active site hydr oxyl of gamma GTase to form a covalent, tetrahedral adduct that resembles t etrahedral transition states and intermediates that occur along the reactio n pathway for gamma GTase-catalyzed reactions. Our studies demonstrate that gamma -boroGlu is a competitive inhibitor of the gamma GTase-catalyzed hyd rolysis of gamma Glu-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 uncompeti tive inhibitor of Gly-Gly-promoted transamidation of gamma Glu-AMC. This ob servation is consistent with the kinetic mechanism we determined for gamma GTase-catalyzed transamidation of gamma Glu-AMC by Gly-Gly to form gamma Gl u-Gly-Gly. To probe rate-limiting transition states for gamma GTase catalys is and inhibition, we determined solvent deuterium isotope effects. Solvent isotope effects on k(c)/K-m for hydrolysis of gamma Glu-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 fluc tuation of the initially formed E-S or E-I complex. In contrast, the solven t 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 gamma GTase with gamma Glu-AMC. Thus, the magnitude of this isotope effect suggests the for mation of a catalytically important protonic bridge in the rate-limiting tr ansition state for deacylation.