Pre‐steady‐state kinetic studies of rat kidney γ‐glutamyl transpeptidase confirm its ping‐pong mechanism

Abstract

AbstractThe enzyme γ‐glutamyl transpeptidase (GGT) is implicated in cellular detoxification, the biosynthesis of leukotrienes and control of the physiological concentration of glutathione. It also plays important roles in Parkinson's disease, diabetes, apoptosis inhibition and cancer drug resistance. It catalyses the breakdown of its in vivo substrate, glutathione, by cleaving the amide bond between the γ‐glutamyl and the cysteinylglycine moieties. Threonine is proposed to act as the nucleophile of GGT in the formation of the γ‐glutamyl acyl enzyme intermediate during the acylation step. The γ‐glutamyl moiety is then transferred to a primary amine acceptor substrate (an amino acid or dipeptide) or to a water molecule, to form a compound containing a new isopeptide bond or glutamate in the transamidation or hydrolysis reactions, respectively. In spite of the importance of the role of GGT in human physiology, there is a lack of information about the mechanisms of its catalytic reactions, and in particular the nature of the intermediate formed during the acylation step. In order to gain insight into the formation of the acyl enzyme intermediate, different D‐γ‐glutamylanilides substituted in the para position with electron‐withdrawing and electron‐donating groups were used as donor substrates under conditions where water served as acceptor substrate. A Hammett plot with a slope of zero was obtained for the steady‐state hydrolysis reaction for which deacylation is the rate‐ limiting step. To confirm the ping‐pong mechanism, pre‐steady‐state kinetics of this reaction were then performed with the donor substrate D‐γ‐glutamyl‐p‐nitroanilide, which liberates the chromophore p‐nitroaniline. Experiments using a stopped‐ flow spectrometer and a rapid mix–quench apparatus gave biphasic traces with a burst up to ∼65 ms, the amplitude of which corresponds well with the concentration of the enzyme. These burst kinetics were also observed in the presence of L‐methionine at concentrations ∼15‐fold below its KM value, where deacylation would still be rate limiting. These observations are consistent with the formation of an intermediate during the rapid acylation step and support the modified ping‐pong mechanism proposed for GGT‐ mediated hydrolysis and aminolysis. Copyright © 2004 John Wiley & Sons, Ltd.