Studies on yeast nucleoside triphosphate-nucleoside diphosphate transphosphorylase (nucleoside diphosphokinase). IV. Steady-state kinetic properties with thymidine nucleotides (including 3'-azido-3'-deoxythymidine analogues).

Enzyme Pub Date : 1991-01-01 DOI:10.1159/000468859
S A Kuby, G Fleming, T Alber, D Richardson, H Takenaka, M Hamada
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引用次数: 3

Abstract

A study of the steady-state kinetics of the crystalline brewer's yeast (Saccharomyces carlsbergensis) nucleoside diphosphokinase, with the magnesium complexes of the adenine and thymidine nucleotides as reactants, has led to a postulated kinetic mechanism which proceeds through a substituted enzyme. This agrees with the earlier conclusions of Garces and Cleland [Biochemistry 1969; 8:633-640] who characterized a reaction between the magnesium complexes of the adenine and uridine nucleotides. An advantage of using thymidine nucleotides as reactants is that they permit accurate, rapid and continuous assays of the enzymatic activity in coupled-enzymatic tests. Through measurements of the initial velocities and product inhibition studies, the Michaelis constants, maximum velocities, and inhibition constants could be evaluated for the individual substrates. Competitive substrate inhibition was encountered at relatively high substrate concentrations, which also permitted an evaluation of their ability to act as 'dead-end' inhibitors. The Michaelis constants for the 3'-azido-3'-deoxythymidine (AzT) analogues were also evaluated and, although these values were only somewhat higher than those of their natural substrates, the Km's for the adenine nucleotides as paired substrates were lower and the Vmax's were drastically reduced. The pharmacological implications of these observations are touched upon and extrapolated to the cases where therapeutic doses of AzT may be employed.

酵母核苷三磷酸核苷二磷酸转磷酸化酶(核苷二磷酸激酶)的研究。胸腺嘧啶核苷酸(包括3'-叠氮-3'-脱氧胸腺嘧啶类似物)的稳态动力学性质。
以腺嘌呤和胸腺嘧啶核苷酸的镁复合物为反应物,对结晶啤酒酵母(Saccharomyces carlsbergensis)核苷二磷酸激酶的稳态动力学进行了研究,得出了一种假设的动力学机制,该机制通过取代酶进行。这与Garces和Cleland的早期结论一致[生物化学1969;[8:63 -640]他描述了腺嘌呤和尿嘧啶核苷酸的镁复合物之间的反应。使用胸腺嘧啶核苷酸作为反应物的一个优点是,它们允许在偶联酶试验中准确、快速和连续地测定酶的活性。通过测量初始速度和产物抑制研究,可以对单个底物的米切里斯常数、最大速度和抑制常数进行评估。在相对较高的底物浓度下,会遇到竞争性底物抑制,这也允许评估它们作为“死端”抑制剂的能力。对3'-叠氮-3'-脱氧胸腺嘧啶(AzT)类似物的Michaelis常数也进行了评估,虽然这些值仅略高于它们的天然底物,但腺嘌呤核苷酸作为配对底物的Km值较低,Vmax值急剧降低。这些观察结果的药理学意义被触及并推断到可能采用治疗剂量的AzT的情况。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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