Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
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- 作者：Florence Gaucher-Wieczorek ; Vincent Gué ; rineau ; David Touboul ; david.touboul@cnrs.fr" class="auth_mail ; Sophie Thé ; tiot-Laurent ; Franck Pelissier ; Marie-Ange Badet-Denisot ; Bernard Badet ; Philippe Durand
- 关键词：Glucosamine-6P synthase ; MALDI-TOF ; Enzyme assay ; Bisubstrate enzyme
- 刊名：Analytical Biochemistry
- 出版年：1 August 2014
- 年：2014
- 卷：458
- 期：Complete
- 页码：61-65
- 全文大小：953 K
文摘
Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5′-diphospho-N-acetyl-d-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts d-fructose-6-phosphate (Fru-6P) and l-glutamine (Gln) into d-glucosamine-6-phosphate (GlcN-6P) and l-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.