一种用于转酮醇酶的新型磷酸赤藓糖4-脱氢酶偶联测定法。

A new erythrose 4-phosphate dehydrogenase coupled assay for transketolase.

作者信息

Naula Christina, Alibu Vincent P, Brock Janice M, Veitch Nicola J, Burchmore Richard J S, Barrett Michael P

机构信息

University of Glasgow, Institute of Biomedical and Life Sciences, Division of Infection & Immunity, The Glasgow Biomedical Research Centre, Glasgow G12 8TA, UK.

出版信息

J Biochem Biophys Methods. 2008 Apr 24;70(6):1185-7. doi: 10.1016/j.jprot.2007.11.002. Epub 2007 Nov 12.

DOI:10.1016/j.jprot.2007.11.002

PMID:18053578

Abstract

The standard assay for transketolase (E.C 2.2.1.1) has depended upon the use of D-xylulose 5-phosphate as the ketose donor substrate since the production of D-glyceraldehyde 3-phosphate can be readily coupled to a reaction that consumes NADH allowing the reaction to be followed spectrophotometrically. Unfortunately, commercial supplies of D-xylulose 5-phosphate recently became unavailable. In this article we describe the coupling of a transketolase reaction (using Leishmania mexicana transketolase) that converts D-fructose 6-phosphate to D-erythrose 4-phosphate. D-Erythrose 4-phosphate can then be converted to 4-phosphate D-erythronate using erythrose-4-phosphate dehydrogenase (E.C 1.2.1.72), a reaction that reduces NAD+ to NADH and can be easily followed spectrophotometrically. D-Ribose 5-phosphate and D-glyceraldehyde 3-phosphate can both be used as ketol acceptor substrates in the reaction although D-ribose 5-phosphate is also a substrate for the coupling enzyme.

摘要

转酮醇酶（E.C 2.2.1.1）的标准检测方法一直依赖于使用D-木酮糖5-磷酸作为酮糖供体底物，因为3-磷酸-D-甘油醛的生成可以很容易地与一个消耗NADH的反应偶联，从而使该反应能够通过分光光度法进行监测。不幸的是，D-木酮糖5-磷酸的商业供应最近无法获得。在本文中，我们描述了一种转酮醇酶反应（使用墨西哥利什曼原虫转酮醇酶）的偶联，该反应将6-磷酸-D-果糖转化为4-磷酸-D-赤藓糖。然后，4-磷酸-D-赤藓糖可以使用赤藓糖-4-磷酸脱氢酶（E.C 1.2.1.72）转化为4-磷酸-D-赤藓糖酸，该反应将NAD+还原为NADH，并且可以很容易地通过分光光度法进行监测。在该反应中，5-磷酸-D-核糖和3-磷酸-D-甘油醛都可以用作酮醇受体底物，尽管5-磷酸-D-核糖也是偶联酶的底物。