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来自面包酵母的腺苷5'-焦磷酸硫酸化酶的纯化及稳态动力学

Purification and steady-state kinetics of adenosine 5'-pyrophosphate sulphurylase from baker's yeast.

作者信息

Nicholls R G

出版信息

Biochem J. 1977 Jul 1;165(1):149-55. doi: 10.1042/bj1650149.

DOI:10.1042/bj1650149
PMID:329837
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1164880/
Abstract

ADP sulphurylase (EC 2.7.7.5) was purified by chromatography on Sephadex G-200 and DEAE-cellulose. The enzyme was assayed by measuring the incorporation of [32P]Pi into ADP in the presence of the substrate for the reverse reaction, adenosine 5'-sulphatophosphate. In the concentration ranges investigated, by using initial-velocity, product-inhibition and isotope-exchange studies, the data were consistent with a Ping Pong reaction mechanism, with Km for adenosine 5'-sulphatophosphate of 1.20 +/- 0.08 mM and a Km for Pi of 4.95 +/- 0.15 mM. Competitive substrate inhibition by Pi (Ki = 11.7 +/- 0.3 mM) was found. ADP sulphurylase catalyses a sulphate-independent Pi-ADP exchange reaction, the kinetics of which are consistent with the kinetics of the overall reaction, inconsistent with the assay of Burnell & Anderson [(1973) Biochem. J. 133, 417-428], which is based on a sulphate-dependent Pi-ADP exchange reaction.

摘要

通过在葡聚糖G - 200和二乙氨基乙基纤维素上进行色谱分离纯化了ADP硫酸化酶（EC 2.7.7.5）。在反向反应的底物腺苷5'-磷酸硫酸存在下，通过测量[32P]Pi掺入ADP的量来测定该酶的活性。在所研究的浓度范围内，通过使用初速度、产物抑制和同位素交换研究，数据与乒乓反应机制一致，腺苷5'-磷酸硫酸的Km为1.20±0.08 mM，Pi的Km为4.95±0.15 mM。发现Pi存在竞争性底物抑制作用（Ki = 11.7±0.3 mM）。ADP硫酸化酶催化一个不依赖硫酸盐的Pi - ADP交换反应，其动力学与总反应的动力学一致，这与Burnell和Anderson [(1973) Biochem. J. 133, 417 - 428]基于依赖硫酸盐的Pi - ADP交换反应的测定方法不一致。

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The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory.具有两种或更多种底物或产物的酶催化反应动力学。II. 抑制作用：命名法与理论
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The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations.具有两种或更多种底物或产物的酶催化反应动力学。I. 命名法和速率方程。
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[Yeast ADP-sulfurylase catalyzing an exchange between orthophosphate and the terminal phosphate of nucleoside diphosphates].[酵母腺苷二磷酸硫酸化酶催化正磷酸盐与核苷二磷酸末端磷酸之间的交换]
Biochim Biophys Acta. 1966 Jul 20;123(1):1-16.
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A simple apparatus for vertical flat-sheet polyacrylamide gel electrophoresis.一种用于垂直平板聚丙烯酰胺凝胶电泳的简易装置。
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