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 and Dementia with Lewy bodies. 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 simple and cost-effective procedures to study the enzyme behaviour. 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 PNPG. Characterization of the enzyme in human leukocytes for activation parameters (optimal pH, Km, and Vmax) and enzyme inhibition, was done. The results indicate that the optimum pH of GCase enzyme with PNPG is 5.1. The Km and Vmax values were 12.6mM and 333 U/mg, respectively. Gluconolactone successfully inhibits GCase in a competitive manner, with a Ki value of 0.023 mM and IC 50 of 0.047 mM. Glucose inhibition was uncompetitive with a Ki of 1.94 mM and IC50 of 55.3 mM. This is the first report for the inhibitory effect of glucose, δ-gluconolactone on leukocyte GCase activity.