Jin S L, Swinnen J V, Conti M
Department of Pediatrics, University of North Carolina, Chapel Hill 27599.
J Biol Chem. 1992 Sep 15;267(26):18929-39.
Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction. The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells. The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity. After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed. Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed. Further deletions within the amino terminus produced inactive proteins. Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs. Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE. However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine. Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity. In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme. Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins. These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.
在大多数已知的环核苷酸磷酸二酯酶（PDE）序列中，约270个氨基酸的区域存在相当大的结构相似性，这使得该区域编码该酶催化结构域成为可能。为了验证这一假设，通过缺失突变和定点诱变分析了一种高亲和力cAMP PDE（cAMP-PDE）的结构。采用基于聚合酶链反应片段扩增的策略对大鼠PDE3 cDNA进行突变。通过在原核和真核细胞中表达野生型和突变蛋白来确定引入突变的效果。使用两种特异性cAMP-PDE多克隆抗体通过免疫印迹分析并通过测量PDE活性来监测PDE蛋白的表达水平。在去除保守结构域侧翼羧基末端的一个99个氨基酸的区域后，该蛋白保留了其催化活性，尽管其Km和速度发生了变化。该PDE氨基末端的内部缺失表明，当去除一个97个氨基酸的片段（从Tyr49到Lys145）时，酶活性增加。氨基末端的进一步缺失产生无活性的蛋白。在催化似乎必不可少的结构域内，所有PDE中1个苏氨酸和2个丝氨酸残基是保守的。用丙氨酸、脯氨酸或丝氨酸取代最保守区域中存在的不变苏氨酸（Thr349），产生的蛋白大小正确，表达水平与野生型PDE相当。然而，在使用的两种表达系统中，除了苏氨酸被丝氨酸取代外，蛋白完全丧失了水解环核苷酸的能力。相反，催化结构域中存在的另外2个保守丝氨酸残基（Ser305和Ser398）的突变要么没有影响，要么仅在Km和Vmax上产生变化，但没有消除催化活性。此外，靠近Thr349的2个组氨酸残基（His278和His311）似乎对催化结构域的结构至关重要，因为在这些残基上进行的任何取代都会产生无活性的酶。与cAMP依赖性蛋白激酶调节亚基的cAMP结合位点同源区域中的一个丝氨酸残基（Ser295）的突变表明，该区域在这两种蛋白中不具有相同的功能。这些数据提供了直接证据，表明一个37 kDa的结构域，其部分对应于所有PDE中的保守区域，包含催化结构域，并且苏氨酸和组氨酸残基可能参与催化和/或对活性酶的构象至关重要。
