Karatas Mesut, Dogan Senol, Spahiu Emrulla, Ašić Adna, Bešić Larisa, Turan Yusuf
International Burch University, Faculty of Engineering and Natural Sciences, Department of Genetics and Bioengineering, Francuske revolucije bb, 71000 Sarajevo, Bosnia and Herzegovina.
Leipzig University, Faculty for Physics and Earth Sciences, Peter Debye Institute, Linnéstraße 5, 04103, Leipzig, Germany.
Heliyon. 2020 Nov 2;6(11):e05191. doi: 10.1016/j.heliyon.2020.e05191. eCollection 2020 Nov.
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 ()-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 -Nitrophenyl-β-D-glucopyranoside (PNPG). Characterization of the enzyme in human leukocytes for activation parameters (optimal pH, K, and V) and enzyme inhibition was done. The results indicate that the optimum pH of GCase enzyme with PNPG is 5.0. The K and V values are 12.6mM and 333 U/mg, respectively. Gluconolactone competitively inhibits GCase, with a K value of 0.023 mM and IC of 0.047 mM. Glucose inhibition is uncompetitive with a K of 1.94 mM and IC of 55.3 mM. This is the first report for the inhibitory effect of glucose, δ-gluconolactone on human leukocyte GCase activity.
葡糖神经酰胺酶（GCase）是一种溶酶体酶，可催化将葡萄糖脑苷脂（GC）的β-糖苷键裂解为葡萄糖和神经酰胺；因此，在复合脂质的降解和细胞膜的周转中发挥着重要作用。据报道，编码该酶——葡糖神经酰胺酶β酸性1（）的基因中，有460种突变不断增加，这些突变会消除其催化活性并降低其酶稳定性，使其与严重健康状况相关，如戈谢病（GD）、帕金森病（PD）和路易体痴呆（DLB）。尽管野生型葡糖神经酰胺酶的三维结构已被阐明，但对其在人类细胞中的特征知之甚少。此外，在酶治疗疗法中被证明对疾病治疗有效的GCase替代来源，使得需要一种简单且经济高效的程序来研究酶的行为。这项工作首次展示了一种成熟、简单、经济且高效的方案，用于通过人工底物对硝基苯基-β-D-吡喃葡萄糖苷（PNPG）研究人类白细胞中的GCase酶。对人类白细胞中的酶进行了激活参数（最佳pH、K和V）和酶抑制的表征。结果表明，GCase酶与PNPG的最佳pH为5.0。K和V值分别为12.6mM和333U/mg。葡萄糖酸内酯竞争性抑制GCase，K值为0.023mM，IC为0.047mM。葡萄糖抑制是非竞争性的，K为1.94mM，IC为55.3mM。这是关于葡萄糖、δ-葡萄糖酸内酯对人类白细胞GCase活性抑制作用的首次报道。
