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.