动能和光谱表征H178A methionyl氨基肽酶大肠杆菌。
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鲁泽WT Copik AJ, Swierczek SI, D’索萨VM,马修斯BW, Holz RC
动能和光谱表征H178A methionyl氨基肽酶大肠杆菌。
生物化学。2003年5月27日,42 (20):6283 - 92。
- PubMed ID
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12755633 (在PubMed]
- 文摘
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来洞察的作用严格守恒的组氨酸残基,H178,反应机理的methionyl氨肽酶的大肠杆菌(EcMetAP-I) H178A突变酶。Metal-reconstituted H178A只绑定一个相当于公司(II)或铁(II)紧密与WT酶亲和力,是相同的基于动力学和等温滴定量热法(ITC)数据。电子吸收光谱有限公司(II))下载H178A EcMetAP-I表明,活性部位pentacoordinate二价金属离子,与WT酶相同。这些数据表明,金属结合位点并未受到改变H178。改变H178在活动的影响,一般来说,由于减少k (cat)。公司的k (cat)值(II))下载H178A MGMM和减少70倍290倍向MP-p-NA WT酶相比,而k (cat)向MGMM下降50倍的铁(II))下载对MP-p-NA H178A酶和140倍。K (m)值MGMM依然不受影响,而MP-p-NA增加约2倍为有限公司(II)和铁(II))下载H178A。k (cat) / k (m)值有限公司(II)和铁(II))下载H178A向两个底物范围从约50 - 580倍减少。pH值依赖日志K (m),日志K (cat),和日志(K (cat) / K (m)) WT和H178A EcMetAP-I也获得相同,内部错误,H178A和WT EcMetAP-I。因此,H178A催化地催化的重要但不是必需的。 Assignment of one of the observed pK(a) values at 8.1 for WT EcMetAP-I was obtained from plots of molar absorptivity at lambda(max(640)) vs pH for both WT and H178A EcMetAP-I. Apparent pK(a) values of 8.1 and 7.6 were obtained for WT and H178A EcMetAP-I, respectively, and were assigned to the deprotonation of a metal-bound water molecule. The data reported herein provide support for the key elements of the previously proposed mechanism and suggest that a similar mechanism can apply to the enzyme with a single metal in the active site.