Monomeric sarcosine oxidase: role of histidine 269 in catalysis.
Monomeric sarcosine oxidase: role of histidine 269 in catalysis.
复制标题
单体肌氨酸氧化酶：组氨酸 269 的催化作用。
DOI：
10.1021/bi020286f
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Jorns,MarilynSchuman
中科院分区：
文献类型：
--
作者：
Zhao,Gouhua;Song,Hui;Chen,Zhi-Wei;Mathews,FScott;Jorns,MarilynSchuman
His 269的保守突变（Asn、Ala或Gln）不会显著影响单体肌氨酸氧化酶（MSOX）的表达、共价黄素化、结合FAD的理化性质或整体蛋白质结构。然而，与肌氨酸和限制速率的还原半反应与l-脯氨酸在pH 8.0的营业额，近2个数量级慢于野生型MSOX。His 269 Asn与吡咯-2-羧酸盐复合物的晶体结构表明，与野生型MSOX相比，抑制剂的吡咯环被置换。His 269突变体都与吡咯-2-羧酸或甲硫基乙酸形成电荷转移复合物，但与野生型MSOX相比，电荷转移带向更短的波长（更高的能量）移动。野生型MSOX和His 269 Asn突变体都结合两性离子形式的脯氨酸。与His 269 Asn突变体或野生型MSOX形成的Eox·l-脯氨酸复合物含有将两性离子脯氨酸转化为活性阴离子形式所需的可电离基团（pKa= 8.0），表明His 269不是活性位点碱基。我们建议，在His 269突变后观察到的配体取向的变化导致最高占据轨道的配体与黄素的最低未占据轨道的最佳重叠。假设的轨道重叠的影响可能占电荷转移带的能量增加和观察到的电子转移速率较慢的突变体酶复合物与电荷转移配体和基板，分别。
Conservative mutation of His269 (to Asn, Ala, or Gln) does not-significantly affect the expression of monomeric sarcosine oxidase (MSOX), covalent flavinylation, the physicochemical properties of bound FAD, or the overall protein structure. Turnover with sarcosine and the limiting rate of the reductive half-reaction withl-proline at pH 8.0 are, however, nearly 2 orders of magnitude slower than that with with wild-type MSOX. The crystal structure of the His269Asn complex with pyrrole-2-carboxylate shows that the pyrrole ring of the inhibitor is displaced as compared with wild-type MSOX. The His269 mutants all form charge-transfer complexes with pyrrole-2-carboxylate or methylthioacetate, but the charge-transfer bands are shifted to shorter wavelengths (higher energy) as compared with wild-type MSOX. Both wild-type MSOX and the His269Asn mutant bind the zwitterionic form ofl-proline. The Eox·l-proline complex formed with the His269Asn mutant or wild-type MSOX contains an ionizable group (pKa= 8.0) that is required for conversion of the zwitterionicl-proline to the reactive anionic form, indicating that His269 is not the active-site base. We propose that the change in ligand orientation observed upon mutation of His269 results in a less than optimal overlap of the highest occupied orbital of the ligand with the lowest unoccupied orbital of the flavin. The postulated effect on orbital overlap may account for the increased energy of charge-transfer bands and the slower rates of electron transfer observed for mutant enzyme complexes with charge-transfer ligands and substrates, respectively.