L-LACTATE OXIDASE AND L-LACTATE MONOOXYGENASE - MECHANISTIC VARIATIONS ON A COMMON STRUCTURAL THEME
L-LACTATE OXIDASE AND L-LACTATE MONOOXYGENASE - MECHANISTIC VARIATIONS ON A COMMON STRUCTURAL THEME
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DOI：
10.1016/0300-9084(96)88178-8
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发表时间：
1995-01-01
期刊：
影响因子：
3.9
通讯作者：
MASSEY, V
中科院分区：
文献类型：
--
作者：
MAEDAYORITA, K;AKI, K;MASSEY, V
本文报道了绿色气球菌产L乳酸酶的性质。编码该酶的基因已经被分离出来。氨基酸序列与黄色素b(2)、乳酸单加氧酶、乙醇酸氧化酶、扁桃酸脱氢酶和长链α-羟基酸氧化酶等其他催化L-α-羟基酸氧化的酶有很高的相似性。该酶在大肠杆菌中表达，是一种以FMN为辅基的黄素蛋白。它具有其他α-羟基酸氧化酶的许多特性，例如稳定黄素的阴离子半喹酮形式，容易形成黄素-N(5)-亚硫酸盐加合物，以及结合黄素周围一组保守的氨基酸残基。对该酶的稳态反应动力学和快速反应动力学进行了研究，发现该酶与L乳酸单加氧酶有许多相似之处，但在定量方面与后者有所不同。正是这两种酶之间的这些定量差异解释了所催化的整个反应的不同。这些差异是由于一种常见的还原黄素酶和丙酮酸中间体的不同稳定性造成的。在单加氧酶的情况下，该络合物非常稳定，并且是与O-反应生成发生氧化脱羧基的络合物，生成醋酸盐、二氧化碳和H2O(Lockbridge O，Massey V，Sullivan PA(1972)J Biol Chem 247,8097-8106)。在乳酸氧化酶的作用下，络合物迅速解离，结果是酶的自由还原黄素形式与O-2反应，生成观察到的产物丙酮酸和过氧化氢。
Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-alpha-hydroxy acids, including flavocytochrome b(2), lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydroxy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many properties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state- and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable, and is the form that reacts with O-2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O-2, to give the observed products, pyruvate and H2O2.