Determination of the mechanism and kinetic constants for hog kidney γ-glutamyltransferase

Abstract

The initial-velocity kinetics of hog kidney gamma-glutamyltransferase were studied. Glutamate gamma-(4-nitroanilide) and its 3-carboxy derivative, glutamate gamma-(3-carboxy-4-nitroanilide), served as gamma-glutamyl donors, and glycylglycine as an acceptor. Reaction products were identified by paper chromatography and amino acid analysis. Inhibited Ping Pong mechanisms and a comprehensive initial- velocity expression were developed which account for the observed simultaneous gamma-glutamyl transfer and autotransfer, competitive inhibition by glycylglycine, and non-competitive inhibition by the carboxy donor. The validity of the proposed Ping Pong mechanisms are supported by enzyme-velocity data obtained with constant ratios of acceptor to donor concentrations. Kinetic constants were determined by a non-linear regression analysis. With glutamate gamma-(4-nitroanilide) as the donor, Michaelis constants for the donor, acceptor and donor-acting-as-acceptor are 1.87, 24.9, and 2.08 mM respectively. With glutamate gamma-(3-carboxy-4-nitroanilide) as the donor, these Michaelis constants are 1.63, 16.6, and 12.3 mM. Glyclyglycine competitive inhibition constants with the parent donor and its carboxy derivative are 275 and 205 mM respectively; the non-competitive inhibition constant of the carboxy donor is 34 mM.

Kinetics of chymotrypsin- and papain-catalysed synthesis of [leucine]enkephalin and [methionine]enkephalin

“…2, inset a). Unlike the lines for a simple ping-pong mechanism, the lines did not converge on the vertical axis but to the left of it, as could be expected from a modified ping-pong mechanism (London et al, 1976).…”

Kinetic analysis of γ-glutamyltransferase reaction process for measuring activity via an integration strategy at low concentrations of γ-glutamyl p-nitroaniline

“…Assigning the instantaneous absorbance in a reaction curve under analysis to A, the maximal absorbance to A m , the absorptivity of p-nitroaniline to ε, glycinylglycine concentration to c A , GGT K m for GGPNA to D m K , and the competitive inhibition constant of glycinylglycine on GGT to A i , K the differential rate equation for GGT reaction was integrated into Eq. (1) (see Appendix) (London et al, 1976):…”

“…(1) K potentially had great effects on V m and required optimizations while other kinetic parameters were those for hog kidney GGT (London et al, 1976). After V m was estimated, the PSC was fixed at 0.11 mmol/L to calculate the initial rate (V i ) from V m by multiplying V m with the CF defined in Eq.…”

“…1) (Liao et al, 2009;Liu et al, 2009). GGT from different sources showed different kinetic parameters (London et al, 1976;Cook and Peters, 1986;Cook et al, 1987;Dvorakova et al, 1996;Stein et al, 2001;Castonguay et al, 2007). Hence, using 2-s intervals to monitor GGT reaction curves within 10 min to enhance confidence in achieving an overlapped region of initial rates measurable by the two individual methods, K , or vice versa, should be optimized for kinetic analyses of GGT reaction curves to estimate initial rates so that the integration strategy can be validated.…”

“…Measurement of GGT with GGPNA manifests the disadvantages of the classical initial rate method because GGPNA has low solubility, GGT has low affinity for GGPNA, and there are inhibitions of GGT by both substrates and products (London et al, 1976;Cook and Peters, 1986;Cook et al, 1987;Dvorakova et al, 1996;Stein et al, 2001;Castonguay et al, 2007). Diversified reactions of GGT on GGPNA and inhibitions of GGT by GGPNA and products complicate the kinetics of the GGT reaction on GGPNA, which results in difficulty in establishing the improved integrated method for the GGT reaction.…”