Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES)

Abstract

The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or gamma-glutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a K(i) of approx. 3 microM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.

Figure 1. Structure of BPTES

Figure 2. Specificity of BPTES inhibition

The activities of KGA, LGA, glutamyl dehydrogenase (GDH) and γ-glutamyl transpeptidase (gGTP) were assayed in the presence and absence of 10 μM BPTES. The assays were performed in triplicate and the results represent the mean of the percentage activity remaining±S.D.

Figure 3. BPTES inhibition with respect to glutamine

(A) Glutamine saturation profiles for rKGA_Δ1 in the absence (●) or presence of 1 μM (■) or 5 μM (▲) BPTES. Enzymatic activity is plotted as μmol/min per ml against the glutamine concentration. Saturation profiles represent the non-linear least-squares fit to the Michaelis–Menten equation. Error bars represent the S.E.M. for quadruplicate activity measurements at each glutamine concentration. (B) Lineweaver–Burk double-reciprocal representation of the glutamine saturation profiles for rKGA_Δ1 in the absence (●) or presence of 1 μM (■) or 5 μM (▲) BPTES. Lines represent a linear least-squares fit of the mean activity measured at each glutamine concentration.

Figure 4. BPTES inhibition with respect to phosphate

Phosphate activation profiles for rKGA_Δ1 in the absence (●) or presence of 1 μM (■) and 3 μM (▲) BPTES. The inset plot is an enlargement of the data obtained at low phosphate concentrations. Enzymatic activity is plotted as μmol/min per ml against the phosphate concentration. Activation curves represent the non-linear least-squares fit of the data to the Hill equation. Error bars represent the S.E.M. for triplicate activity measurements at each phosphate concentration.

Figure 5. Gel-filtration profiles of rKGA_Δ1 in the presence or absence of BPTES

As indicated, 25 μl samples of rKGA_Δ1 in the absence (broken line) or presence of 10 μM BPTES (continuous line) were injected on to a Bio-Silect column equilibrated with buffer containing 10 mM (A) or 200 mM phosphate (B). Protein elution was monitored at A₂₃₀.

Figure 6. Sedimentation-velocity analysis of rKGA_Δ1

Data are presented as G(s) plots of the integral distribution of s_20,w against boundary fraction (%). (A) Concentration-dependent analysis using 0.8, 2.5 and 15 μM rKGA_Δ1 in the presence of 10 mM phosphate. (B) Phosphate-dependent analysis using 2.5 μM rKGA_Δ1 samples in buffers containing 0, 50 or 200 mM phosphate.

Figure 7. Analytical-ultracentrifugation analysis of rKGA_Δ1 in the presence of 10 μM BPTES

(A) Sedimentation-velocity analysis of 2.5 μM rKGA_Δ1 in the presence of 10 μM BPTES in buffers containing 10 or 200 mM phosphate. (B) Sedimentation-equilibrium analysis of 12 scans taken at three loading concentrations and multiple speeds were fitted to a single-component non-interaction ideal model. The left panel contains data obtained from the analysis of rKGA_Δ1 in 10 mM phosphate and the right panel are data obtained in 200 mM phosphate buffer. Overlays are shown at the bottom of each panel and the residual plot of the fit is shown in the top of each panel. The variance for the fits to a single-component non-interacting ideal species model was 3.6×10⁻⁵ and 6.5×10⁻⁵ respectively. Optical density is equivalent to attenuance (D).