Enzyme kinetics and inhibition parameters of human leukocyte glucosylceramidase

Abstract

Glucosylceramidase (GCase) is a lysosomal enzyme that catalyzes the cleavage of β-glucosidic linkage of glucocerebroside (GC) into glucose and ceramide; thereby, plays an essential function in the degradation of complex lipids and the turnover of cellular membranes. The growing list of 460 mutations in the gene coding for it-glucosylceramidase beta acid 1 (GBA1)-is reported to abolish its catalytic activity and decrease its enzyme stability, associating it with severe health conditions such as Gaucher disease (GD), Parkinson Disease (PD) and Dementia with Lewy bodies (DLB). Although the three-dimensional structure of wild type glucosylceramidase is elucidated, little is known about its features in human cells. Moreover, alternative sources of GCase that prove to be effective in the treatment of diseases with enzyme treatment therapies, impose the need for a simple and cost-effective procedure to study the enzyme behavior. This work, for the first time, shows a well-established, yet simple, cost- and time-efficient protocol for the study of GCase enzyme in human leukocytes by the artificial substrate p-Nitrophenyl-β-D-glucopyranoside (PNPG). Characterization of the enzyme in human leukocytes for activation parameters (optimal pH, K_m, and V_max) and enzyme inhibition was done. The results indicate that the optimum pH of GCase enzyme with PNPG is 5.0. The K_m and V_max values are 12.6mM and 333 U/mg, respectively. Gluconolactone competitively inhibits GCase, with a K_i value of 0.023 mM and IC₅₀ of 0.047 mM. Glucose inhibition is uncompetitive with a K_i of 1.94 mM and IC₅₀ of 55.3 mM. This is the first report for the inhibitory effect of glucose, δ-gluconolactone on human leukocyte GCase activity.

Keywords: Biochemistry; Clinical research; Enzyme kinetics; Gaucher disease; Gluconolactone; Glucose; Glucosylceramidase; Glucosylceramide; Hematological system; Human leukocyte; Inhibition parameters; Molecular biology; PNPG; Proteins.

Figure 1

Glucosylceramidase (GCase) enzyme stability in human leukocyte homogenate stored at -20 °C. Enzyme activity at day zero was taken as 100%, and the remaining results were normalized according to this value.

Figure 2

(A) The optimal pH for human leukocyte Glucosylceramidase (GCase) activity is 5.0. The influence of varying pH values on the activity was determined by using sodium acetate buffer in the pH range 4.0–5.5. (B) Lineweaver-Burk plot for Michaelis constant (K_m) determination of GCase at different p-nitrophenyl-β-D-glucopyranoside (PNPG) substrate concentrations (0.71 mM–2.50 mM) and in sodium acetate buffer (50mM, pH 5.0), reveals a K_m of 12.6 mM and V_max of 333 U/mg. All samples were assayed in the presence of 6 mg/ml sodium taurocholate and 70 μl enzyme solution.

Figure 3

(A) The plot of relative enzyme activity (%) of Glucosylceramidase (GCase) in different concentrations of δ-gluconolactone (B) Lineweaver–Burk plot analysis of GCase enzyme, inhibited by the increasing concentration of δ-gluconolactone. GCase activity was measured with p-nitrophenyl-β-D-glucopyranoside (PNPG) as substrate in the absence or presence of δ-gluconolactone (Inhibitor 1 with 0.0128 mM and Inhibitor 2 with 0.0642 mM). The intercept of the plots indicates competitive inhibition by δ-gluconolactone (See Figure 5).

Figure 4

(A) The relative activity (%) curve of the Glucosylceramidase (GCase) in the presence of different glucose concentrations (B) Lineweaver–Burk plot analysis of the GCase inhibited by increasing the concentration of glucose. GCase activity was measured with p-nitrophenyl-β-D-glucopyranoside (PNPG) as the substrate, in the absence or presence of glucose concentrations (Inhibitor 1 with 5.07 mM and Inhibitor 2 with 62.8 mM). Glucose inhibits the enzyme in an uncompetitive manner (See Figure 5).

Figure 5

The intersection points of Lineweaver–Burk plots. The increasing concentration of GCase inhibition by (A) δ-gluconolactone (B) glucose.