Gamma-glutamyl hydrolase: kinetic characterization of isopeptide hydrolysis using fluorogenic substrates

Abstract

Gamma-glutamyl hydrolase, a cysteine peptidase, catalyzes the hydrolysis of poly-gamma-glutamate derivatives of folate cofactors and many antifolate drugs. We have used internally quenched fluorogenic derivatives of glutamyl-gamma-glutamate and (4,4-difluoro)glutamyl-gamma-glutamate to examine the effect of fluorine substitution adjacent to the scissile isopeptide bond. Using a newly developed continuous fluorescence assay, the hydrolysis of both substrates could be described by Michaelis-Menten kinetics. Fluorine substitution resulted in a significant decrease in observed rates of hydrolysis under steady-state conditions due primarily to a approximately 15-fold increase in Km. Using stopped-flow techniques, hydrolysis of the non-fluorinated isopeptide was characterized by a burst phase followed by a steady-state rate, indicating that formation of the acyl enzyme is not rate-limiting for hydrolysis of this isopeptide. This conclusion was confirmed by analysis of the progress curves over a wide range of substrate concentration, which demonstrated that the acylation rate (k2) is approximately 10-fold higher than the deacylation rate (k3). The increased value of Km associated with the difluoro derivative limited the ability to obtain comparable pre-steady-state kinetics data at saturating concentration of substrate due to inner filter effects. However, even under nonsaturating conditions, a modest burst was observed for the difluoro derivative. These data indicate that either deacylation or rearrangement of the enzyme-product complex is rate-limiting in this isopeptide hydrolysis reaction.

Figure 1

Folylpolyglutamate synthesis and hydrolysis. The folylmonoglutamate is elongated in an ATP-dependent reaction catalyzed by FPGS. GH catalyzes the hydrolysis of the γ-glutamyl bonds.

Figure 2

GH-catalyzed hydrolysis of internally quenched fluorescent substrates 1 and 2.

Figure 3

Proposed kinetic scheme for GH. ES is the Michaelis complex and ES′ is the acyl enzyme.

Figure 4

(A) Burst observed for rapid mixing of Abz-Glu-γ-Glu-γ-Tyr(NO₂) (1, 50 μM) in assay buffer, pH 6.0, and varying concentrations of GH, in enzyme storage buffer, pH 5.5, 4 °C. Final concentrations were 50 μM 1 and 0.29 – 1.2 μM GH as indicated. Each progress curve is the average of five experimental traces. Only 10% of the data points are shown for clarity and each line represents a fit to the burst equation (eq 1). (B) Dependence of the burst amplitude (○) and steady state velocity (■) on enzyme concentration.

Figure 5

Transient kinetics with varying Abz-Glu-γ-Glu-γ-Tyr(NO₂), 1, in assay buffer, pH 6.0, mixed with GH, (0.86 μM final concentration after mixing) in enzyme storage buffer, pH 5.5, 4 °C. (A) Burst kinetics observed by fluorescence at > 389 nm upon varying concentration of 1 (indicated on figure). Each progress curve is the average of at least three experimental traces and only 10% of the data are shown for clarity. Data for [1] > 2.5 μM were fit to eq. 1 (solid lines). Data for 1 > 2.5 μM were fit to eq 1. (B) Dependence of burst rate constant on substrate concentration with fit to eq 2. (C) Dependence of burst amplitude on substrate concentration with fit to eq 3. (D) Dependence of steady-state rate on substrate concentration with fit to the Michaelis-Menten equation.

Figure 6

Kinetics of Abz-4,4-F₂Glu-γ-Glu-γ-Tyr(NO₂) (2) hydrolysis. Equal volumes of GH in storage buffer (pH 5.5) and 2 in assay buffer (pH 6) were rapidly mixed at 4 °C and the fluorescence monitored. Concentrations after mixing were 0.86 μM GH and 150 μM 2, average traces (n=5) shown.

Figure 7

Scheme 1

Scheme 2