Identification of essential residues in 2′,3′-cyclic nucleotide 3′-phosphodiesterase -: Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity
Identification of essential residues in 2′,3′-cyclic nucleotide 3′-phosphodiesterase -: Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity
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DOI：
10.1074/jbc.m009434200
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发表时间：
2001-05-04
影响因子：
4.8
通讯作者：
Braun, PE
中科院分区：
文献类型：
--
作者：
Lee, J;Gravel, M;Braun, PE
2',3'-环核苷酸3'-磷酸二酯酶（CNP; EC 3.1.4.37）在体外催化2',3'-环核苷酸中3'-磷酸二酯键的水解，只产生2'-核苷酸。重组大鼠CNP1三分之二的c端n端缺失图谱发现了一个具有催化结构域的区域，进一步的截断消除了活性。蛋白质水解和动力学分析表明，该结构域形成致密的球状结构，并包含所有催化基本特征。随后，CNP1的催化片段（CNP-CF）被用于化学修饰研究，以确定活性必需的氨基酸残基。对半胱氨酸CNP-CF突变体的5,5′-二硫代比斯-（2-硝基苯甲酸）修饰研究和动力学分析表明，半胱氨酸在酶活性中起非必需作用。另一方面，用焦碳酸二乙酯修饰的研究表明，两种组氨酸是CNPase活性所必需的。结果，仅有的两个保守的组氨酸，His-230和His-309突变为苯丙氨酸和亮氨酸。4个组氨酸突变体的k（cat）值均比野生型CNP-CF低1000倍，但k -m值相似。圆二色性研究表明，组氨酸突变体的低催化活性不是由于二级结构的明显变化。综上所述，这些结果表明两种组氨酸在催化中都起着关键作用。
2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC 3.1.4.37) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K-m values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis.