Arginine 177 is involved in Mn(II) binding by manganese peroxidase
Arginine 177 is involved in Mn(II) binding by manganese peroxidase
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DOI：
10.1021/bi990943c
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发表时间：
1999-08-31
期刊：
影响因子：
2.9
通讯作者：
Gold, MH
中科院分区：
文献类型：
--
作者：
Gelpke, MDS;Moënne-Loccoz, P;Gold, MH
对黄孢原毛平革菌锰过氧化物酶同工酶1（mnp 1）基因进行了定点突变R177 A和R177 K。在初级代谢生长期间，在甘油醛-3-磷酸脱氢酶基因启动子的控制下，在黄孢原毛平革菌中表达突变酶，纯化至均一，并通过光谱和动力学方法表征。铁和氧化态的紫外-可见光谱和铁凝视的共振拉曼光谱与野生型酶的相似，表明血红素环境没有受到Arg 177突变的显著影响。Mn-II的表观K-m值类似于R177 A和R177 K MnP的20倍，比野生型MnP大。然而，表观K-m值的底物，H2 O2和亚铁氰化物，和Mn-II和亚铁氰化物氧化的k（猫）值是类似的野生型酶。化合物I（MnPI）通过Mn-II还原突变体MNP的二级速率常数类似于野生型MNP的10倍。此外，从Mn-II还原MnP化合物II（MnPII）的一阶图计算的突变酶的K-D值类似于野生型MnP的22倍。与此相反，MnPII还原Mn-II的一阶速率常数是相似的突变体和野生型MNP。此外，二阶速率常数的野生型和突变体酶的MnPI形成，MnPI还原溴，和MnPI和MnPII还原亚铁氰化物没有显着变化。这些结果表明，R177 A和R177 K突变具体影响锰的结合，而从Mn-II到氧化血红素的电子转移速率显然不受影响。
Site-directed mutations R177A and R177K in the gene encoding manganese peroxidase isozyme 1 (mnp1) from Phanerochaete chrysosporium were generated. The mutant enzymes were expressed in P. chrysosporium during primary metabolic growth under the control of the glyceraldehyde-3-phosphate dehydrogenase gene promoter, purified to homogeneity, and characterized by spectroscopic and kinetic methods. The UV-vis spectra of the ferric and oxidized states and resonance Raman spectra of the ferric stare were similar to those of the wild-type enzyme, indicating that the heme environment was not significantly affected by the mutations at Arg177. Apparent K-m values for Mn-II were similar to 20-fold greater for the R177A and R177K MnPs than for wild-type MnP. However, the apparent K-m values for the substrates, H2O2 and ferrocyanide, and the k(cat) values for Mn-II and ferrocyanide oxidation were similar to those of the wild-type enzyme. The second-order rate constants for compound I (MnPI) reduction of the mutant MnPs by Mn-II were similar to 10-fold lower than for wild-type MnP. In addition, the K-D values calculated from the first-order plots of MnP compound II (MnPII) reduction by Mn-II for the mutant enzymes were similar to 22-fold greater than for wild-type MnP. In contrast, the first-order rate constants for MnPII reduction by Mn-II were similar for the mutant and wild-type MnPs. Furthermore, second-order rate constants for the wild-type and mutant enzymes for MnPI formation, for MnPI reduction by bromide, and for MnPI and MnPII reduction by ferrocyanide were not significantly changed. These results indicate that both the R177A and R177K mutations specifically affect the binding of Mn, whereas the rate of electron transfer from Mn-II to the oxidized heme apparently is not affected.