Department of Biochemistry and Microbiology, Rhodes University, Makhanda (Grahamstown), South Africa.
Malawi Adventist University, Malamulo Campus, Department of Biomedical Sciences, Makwasa, Malawi.
Int J Nanomedicine. 2023 Mar 23;18:1399-1411. doi: 10.2147/IJN.S401319. eCollection 2023.
Regardless of the efforts to ease cases of human African trypanosomiasis (HAT), an increased number of cases get reported annually. This is because of drug resistant (Tb), the causative agent of the illness. This has renewed the need for creative methods to find new anti-trypanosomal drugs. The blood stream form (BSF) of the parasite depends exclusively on the glycolytic pathway for energy production while it is in the human host. Interruptions in this pathway efficiently kills the parasite. hexokinase (HK) is the first enzyme in glycolysis, and any effectors or inhibitors of HK would have potential as anti-trypanosomal agents.
HK and human glucokinase (GCK) were over-expressed with a 6 histidine-tag in BL21(DE3) cells having the pRARE2 plasmid.
HK had thermal and pH stability between 30°C and 55°C and 7.5 and 8.5, respectively, while GCK exhibited thermal and pH stability between 30°C and 40°C and 7.0 and 8.0, respectively. Kinetically, HK had a K of 39.3 µM, V of 0.066 µmol.min.mL, k of 2.05 min and k/K of 0.0526 min.µmol. GCK exhibited a K of 4.5 µM, V of 0.032 µnmol.min.mL, k of 11.25 min, and k/K of 2.5 min.µmol. Interaction kinetic studies of silver nanoparticles (AgNPs) (0.1 µM) of average size of 6 nm with HK and GCK were conducted. AgNPs selectively inhibited HK over GCK. HK showed a non-competitive inhibition with a 50% and 28% decrease in V, and k/k, respectively. GCK showed a 33% increase in affinity, 9% decrease in V, and a 50% increase in enzyme efficiency.
The observed pattern of hGCK and AgNPs falls under the uncompetitive inhibition. The observed highly selective inhibitory effects of AgNPs between HK and GCK may be used in development of new anti-trypanosomal drugs.
尽管人们努力缓解非洲人类锥虫病（HAT）病例，但每年报告的病例数量仍在增加。这是因为疾病的病原体（Tb）具有耐药性。这就需要重新寻找创造性的方法来寻找新的抗锥虫药物。寄生虫的血液形式（BSF）在人类宿主中完全依赖糖酵解途径来产生能量。该途径的中断可以有效地杀死寄生虫。己糖激酶（HK）是糖酵解途径中的第一个酶，任何 HK 的效应物或抑制剂都可能成为抗锥虫药物。
用 6 个组氨酸标签在含有 pRARE2 质粒的 BL21（DE3）细胞中过表达 HK 和人葡萄糖激酶（GCK）。
HK 的热稳定性和 pH 值稳定性分别在 30°C 至 55°C 和 7.5 至 8.5 之间，而 GCK 的热稳定性和 pH 值稳定性分别在 30°C 至 40°C 和 7.0 至 8.0 之间。动力学方面，HK 的 K 为 39.3 µM，V 为 0.066 µmol.min.mL，k 为 2.05 min，k/K 为 0.0526 min.µmol。GCK 的 K 为 4.5 µM，V 为 0.032 µnmol.min.mL，k 为 11.25 min，k/K 为 2.5 min.µmol。对平均粒径为 6nm 的银纳米粒子（AgNPs）（0.1 µM）与 HK 和 GCK 的相互作用动力学进行了研究。AgNPs 选择性地抑制了 HK 而不是 GCK。HK 表现出非竞争性抑制，V 和 k/K 分别降低了 50%和 28%。GCK 的亲和力增加了 33%，V 降低了 9%，酶效率增加了 50%。
观察到的 hGCK 和 AgNPs 的模式属于非竞争性抑制。AgNPs 在 HK 和 GCK 之间表现出高度选择性的抑制作用，可用于开发新的抗锥虫药物。
