Yavorska Oksana A, Syriste Lukas, du Plessis Chantal M, Yaqoob Maryam, Loogman Kyle, Cordara Michael, Chik John K
Department of Chemistry and Physics, Mount Royal University, Calgary, Alberta T2N4N1, Canada.
Microbiology, Immunology & Infectious Diseases, Cumming School of Medicine, University of Calgary, HSC B724-E, 3330 Hospital Drive NW, Calgary, Alberta T3E 6K6, Canada.
ACS Omega. 2021 Sep 30;6(40):26239-26250. doi: 10.1021/acsomega.1c03243. eCollection 2021 Oct 12.
Examining the effects of different cosolutes on enzyme kinetics yielded glimpses into their potential behavior when functioning in their natural, complex, milieu. Viewing cosolute influences on a model enzyme, calf intestinal alkaline phosphatase, as a combination of competitive and uncompetitive behaviors provided quantitative insights into their effects on catalysis. Observed decreases in the apparent specificity constant, , caused by the presence of polyethylene glycols or betaine in the reaction solution, indicated interference with enzyme-substrate complex formation. This competitive inhibition appeared to be driven by osmotic stress. Dextran 6 K and sucrose strongly impeded the subsequent conversion of the bound substrate into a free product, which was marked by sharp reductions in , uncompetitive inhibition. For the same step, smaller noncarbohydrate cosolutes, triethylene glycol, polyethylene glycol 400, and betaine, also behaved as uncompetitive inhibitors but to a lesser extent. However, polyethylene glycol 8000 and 20,000 were uncompetitive activators, increasing . Polyethylene glycol of molecular weight 1000 displayed intermediate effects between these two groups of noncarbohydrate cosolutes. These results suggested that crowding has a strong influence on free product formation. The combination of competitive and uncompetitive effects and mixed behaviors, caused by the cosolutes on calf intestinal alkaline phosphatase kinetics, was consistent with the trends seen in similar enzyme-cosolute studies. It is proposed that the double-displacement mechanism of alkaline phosphatases, shared by many other enzymes, could be the root of this general observation.
研究不同共溶质对酶动力学的影响，使我们得以初步了解它们在天然复杂环境中发挥作用时的潜在行为。将共溶质对模型酶——小牛肠碱性磷酸酶的影响视为竞争性和非竞争性行为的组合，为深入了解它们对催化作用的影响提供了定量依据。反应溶液中聚乙二醇或甜菜碱的存在导致表观特异性常数（ ）降低，这表明其对酶 - 底物复合物的形成产生了干扰。这种竞争性抑制似乎是由渗透压引起的。右旋糖酐6K和蔗糖强烈阻碍了结合底物随后转化为游离产物，其表现为（ ）急剧下降，即非竞争性抑制。对于同一步骤，较小的非碳水化合物共溶质，如三甘醇、聚乙二醇400和甜菜碱，也表现为非竞争性抑制剂，但程度较轻。然而，聚乙二醇8000和20000却是非竞争性激活剂，会增加（ ）。分子量为1000的聚乙二醇在这两组非碳水化合物共溶质之间表现出中间效应。这些结果表明，拥挤效应（crowding）对游离产物的形成有很大影响。共溶质对小牛肠碱性磷酸酶动力学产生的竞争性和非竞争性效应以及混合行为，与类似酶 - 共溶质研究中观察到的趋势一致。有人提出，许多其他酶所共有的碱性磷酸酶的双置换机制可能是这一普遍现象的根源。
