Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase*
Asp-170 Is Crucial for the Redox Properties of Vanillyl-alcohol Oxidase*
复制标题
Asp-170 对于香草醇氧化酶的氧化还原特性至关重要*
DOI：
10.1074/jbc.275.20.14799
复制
发表时间：
2000
期刊：
影响因子：
--
通讯作者：
W. V. van Berkel
中科院分区：
文献类型：
--
作者：
R. V. D. van den Heuvel;M. Fraaije;A. Mattevi;W. V. van Berkel
香草醇氧化酶是一种含有共价黄素的黄素蛋白，其催化4-（甲氧基甲基）苯酚氧化为4-羟基苯甲醛。该反应通过形成对苯醌甲基化物中间体进行，之后进行加水。Asp-170位于黄素的N5-原子附近，已被提出作为活性位点碱基。为了验证这一假设，我们已经解决了D170 E，D170 S，D170 A和D170 N变体的特性。光谱和荧光分析以及D170 S的晶体结构表明，Asp-170置换不会引起重大结构变化。然而，在D170 A和D170 N中，分别有50%和100%的黄素是非共价结合的。香草醇氧化酶变体的动力学表征表明，催化需要Asp-170。D170 E的活性比野生型酶低50倍，而其他Asp-170变体的活性比野生型酶低约103倍。Asp-170变体的催化作用受损是由缓慢的黄素还原引起的。此外，突变蛋白质已经失去了在还原酶和对苯醌甲基化物中间体之间形成稳定复合物的能力。与野生型香草醇氧化酶（+55 mV）相比，D170 E（+6 mV）和D170 S（-91 mV）中的氧化还原中点电位显著降低。这支持了Asp-170与还原辅因子的质子化N5原子相互作用，从而增加FAD氧化还原电位的观点。综上所述，我们得出结论，天冬氨酸-170参与的过程中的自催化黄素化，是至关重要的有效的氧化还原催化。
Vanillyl-alcohol oxidase is a flavoprotein containing a covalent flavin that catalyzes the oxidation of 4-(methoxymethyl)phenol to 4-hydroxybenzaldehyde. The reaction proceeds through the formation of a p-quinone methide intermediate, after which, water addition takes place. Asp-170, located near the N5-atom of the flavin, has been proposed to act as an active site base. To test this hypothesis, we have addressed the properties of D170E, D170S, D170A, and D170N variants. Spectral and fluorescence analysis, together with the crystal structure of D170S, suggests that the Asp-170 replacements do not induce major structural changes. However, in D170A and D170N, 50 and 100%, respectively, of the flavin is non-covalently bound. Kinetic characterization of the vanillyl-alcohol oxidase variants revealed that Asp-170 is required for catalysis. D170E is 50-fold less active, and the other Asp-170 variants are about 103-fold less active than wild type enzyme. Impaired catalysis of the Asp-170 variants is caused by slow flavin reduction. Furthermore, the mutant proteins have lost the capability of forming a stable complex between reduced enzyme and the p-quinone methide intermediate. The redox midpoint potentials in D170E (+6 mV) and D170S (–91 mV) are considerably decreased compared with wild type vanillyl-alcohol oxidase (+55 mV). This supports the idea that Asp-170 interacts with the protonated N5-atom of the reduced cofactor, thus increasing the FAD redox potential. Taken together, we conclude that Asp-170 is involved in the process of autocatalytic flavinylation and is crucial for efficient redox catalysis.
登录
查看更多内容
人中链酰基辅酶A脱氢酶的氧化还原特性，通过带电荷的活性位点氨基酸残基进行调节。
DOI：
10.1021/bi981414w
发表时间：
1998
期刊：
Biochemistry.
影响因子：
--
作者：
Mancini-Samuelson,GJ;Kieweg,V;Sabaj,KM;Ghisla,S;Stankovich,MT
通讯作者：
Stankovich,MT
影响因子：
4.7
作者：
Bodell, WJ;Ye, QP;Pongracz, K
通讯作者：
Pongracz, K
影响因子：
5.7
作者：
P. Trickey;M. Wagner;M. Jorns;F. Mathews
通讯作者：
P. Trickey;M. Wagner;M. Jorns;F. Mathews
对甲酚甲基羟化酶将 4-乙基苯酚羟基化产生 1-(4-羟基苯基)乙醇的立体化学。
DOI：
10.1042/bj2240617
发表时间：
1984
期刊：
The Biochemical journal
影响因子：
--
作者：
McIntire,W;Hopper,DJ;Craig,JC;Everhart,ET;Webster,RV;Causer,MJ;Singer,TP
通讯作者：
Singer,TP
影响因子：
5.1
作者：
Bolton, JL;Turnipseed, SB;Thompson, JA
通讯作者：
Thompson, JA