Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state
Tyr275 and Lys279 stabilize NADPH within the catalytic site of NADPH:protochlorophyllide oxidoreductase and are involved in the formation of the enzyme photoactive state
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DOI：
10.1021/bi0105025
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发表时间：
2001-10-23
期刊：
影响因子：
2.9
通讯作者：
Timko, MP
中科院分区：
文献类型：
--
作者：
Lebedev, N;Karginova, O;Timko, MP
对异源表达的高纯度重组豌豆NADPH：原叶绿素氧化还原酶(POR)进行了光化学活性、辅因子和底物结合的荧光光谱和动力学分析以及酶的变性研究。用底物的单个立体异构体[C8-乙基-C13(2)-(R)-原叶丙内酯]得到的结果表明，酶的光活性状态在646 nm处具有特征荧光最大值，这是由于酶催化部位存在特定的带电氨基酸所致。在涉及辅因子(NADPH)的直接氢转移的反应中，光活性状态被直接转换为具有685 nm荧光的中间体。对酶催化口袋中高度保守的Tyr275(Y275F)和Lys279(K279I和K279R)残基进行的定点突变表明，野生型POR中这两个氨基酸的存在显著增加了辅因子与底物结合后形成光活性状态的可能性。同时，这两种氨基酸的存在破坏了POR的稳定性，增加了酶的变性速度。Tyr275和Lys279在底物或辅因子与酶的结合中都不起关键作用。此外，Tyr275的存在对于原叶绿素还原反应的第二步是绝对必要的，即685 nm荧光中间体的“暗”转化和最终产物--叶绿素的形成。我们认为Tyr275和Lys279参与了NADPH和PChlide在酶催化部位的适当配位，从而控制了POR光活性状态的形成效率。
Fluorescence spectroscopic and kinetic analysis of photochemical activity, cofactor and substrate binding, and enzyme denaturation studies were performed with highly purified, recombinant pea NADPH: protochlorophyllide oxidoreductase (POR) heterologously expressed in Escherichia coli. The results obtained with an individual stereoisomer of the substrate [C8-ethyl-C13(2)-(R)-protoclilorophyllide] demonstrate that the enzyme photoactive state possesses a characteristic fluorescence maximum at 646 nm that is due to the presence of specific charged amino acids in the enzyme catalytic site. The photoactive state is converted directly into an intermediate having fluorescence at 685 nm in a reaction involving direct hydrogen transfer from the cofactor (NADPH). Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme. At the same time, the presence of these two amino acids destabilizes POR and increases the rate of enzyme denaturation. Neither Tyr275 nor Lys279 plays a crucial role in the binding of the substrate or cofactor by the enzyme. In addition, the presence of Tyr275 is absolutely necessary for the second step of the protochlorophyllide reduction reaction, "dark" conversion of the 685 nm fluorescence intermediate and the formation of the final product, chlorophyllide. We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state.