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荧光假单胞菌的D-葡萄糖-6-磷酸脱氢酶(恩特纳-杜多罗夫酶)。纯化、性质及调控

D-glucose-6-phosphate dehydrogenase (Entner-Doudoroff enzyme) from Pseudomonas fluorescens. Purification, properties and regulation.

作者信息

Lessmann D, Schimz K L, Kurz G

出版信息

Eur J Biochem. 1975 Nov 15;59(2):545-59. doi: 10.1111/j.1432-1033.1975.tb02481.x.

DOI:10.1111/j.1432-1033.1975.tb02481.x
PMID:1257
Abstract
  1. The existence of two different D-glucose-6-phosphate dehydrogenases in Pseudomonas fluorescens has been demonstrated. Based on their different specificity and their different metabolic regulation one enzyme is appointed to the Entner-Doudoroff pathway and the other to the hexose monophosphate pathway. 2. A procedure is described for the isolation of that D-glucose-6-phosphate dehydrogenase which forms part of the Entner-Doudoroff pathway (Entner-Doudoroff enzyme). A 950-fold purification was achieved with an overall yield of 44%. The final preparation, having a specific activity of about 300 mumol NADH formed per min per mg protein, was shown to be homogeneous. 3. The molecular weight of the Entner-Doudoroff enzyme has been determined to be 220000 by gel permeation chromatography, and that of the other enzyme (Zwischenferment) has been shown to be 265000. 4. The pI of the Entner-Doudoroff enzyme has been shown to be 5.24 and that of the Zwischenferment 4.27. The Entner-Doudoroff enzyme is stable in the range of pH 6 to 10.5 and shows its maximal activity at pH 8.9. 5. The Entner-Doudoroff enzyme showed specificity for NAD+ as well as for NADP+ and exhibited homotropic effects for D-glucose 6-phosphate. It is inhibited by ATP which acts as a negative allosteric effector. Other nucleoside triphosphates as well as ADP are also inhibitory. 6. The enzyme catalyzes the transfer of the axial hydrogen at carbon-1 of beta-D-glucopyranose 6-phosphate to the si face of carbon-4 of the nicotinamide ring and must be classified as B-side stereospecific dehydrogenase.
摘要
  1. 已证实在荧光假单胞菌中存在两种不同的6-磷酸-D-葡萄糖脱氢酶。基于它们不同的特异性和不同的代谢调节,一种酶被指定参与恩特纳-杜德洛夫途径,另一种参与磷酸己糖途径。2. 描述了一种分离作为恩特纳-杜德洛夫途径一部分的6-磷酸-D-葡萄糖脱氢酶(恩特纳-杜德洛夫酶)的方法。实现了950倍的纯化,总产率为44%。最终制剂的比活性约为每分钟每毫克蛋白质形成300μmol NADH,显示为均一的。3. 通过凝胶渗透色谱法测定恩特纳-杜德洛夫酶的分子量为220000,另一种酶( Zwischenferment)的分子量已显示为265000。4. 已显示恩特纳-杜德洛夫酶的pI为5.24, Zwischenferment的pI为4.27。恩特纳-杜德洛夫酶在pH 6至10.5范围内稳定,在pH 8.9时显示出最大活性。5. 恩特纳-杜德洛夫酶对NAD+以及NADP+都具有特异性,并且对6-磷酸-D-葡萄糖表现出同向效应。它被作为负别构效应剂的ATP抑制。其他核苷三磷酸以及ADP也具有抑制作用。6. 该酶催化将β-D-吡喃葡萄糖6-磷酸碳-1上的轴向氢转移到烟酰胺环碳-4的si面,必须归类为B面立体特异性脱氢酶。

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