Cheung P P, Yu L, Zhang H, Colman R W
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, 19140, USA.
Arch Biochem Biophys. 1998 Dec 1;360(1):99-104. doi: 10.1006/abbi.1998.0915.
Phosphodiesterases (PDE) are important for the downregulation of the intracellular level of the second messenger cyclic adenosine monophosphate (cAMP) by hydrolyzing cAMP to 5'AMP. Previous studies from our laboratory suggested that the human platelet PDE3A active site has two essential histidine residues and one cysteine residue. We therefore decided to begin mutating histidines and cysteines in the conserved domain of PDE3A. A truncated but catalytically active recombinant PDE3A protein was expressed in yeast cells. A six-histidine tag was engineered to the carboxyl end to facilitate protein purification by nickel column. This cDNA construct, PDE3ADelta1, was used for the mutant construction. Mutations were introduced by mutant oligonucleotides during PCR or DNA replication and were confirmed by sequencing. The mutant cDNAs were expressed in PDE-deficient yeast host Saccharomyces cerevisiae strain GL62. The expression levels of the recombinant PDE mutant proteins were monitored by Western blotting using a rabbit anti-platelet PDE3A polyclonal antibody. The kcat, KM, and IC50 for cyclic guanosine monophosphate (cGMP) and IC50 for milrinone were determined for each mutant. Two highly conserved histidines and four conserved cysteines were each mutated to alanine. C816 is present in the 44-amino acid insert unique to PDE3. The mutant C816A is poorly expressed (>2%) and probably is not folded appropriately. H840 is the second histidine in the second motif for metal binding, HDXXH. The mutation H840A, although expressed moderately well, has undetectable activity and is probably responsible for binding a bivalent cation, e.g., Mn2+, essential for catalysis. H869 is a highly conserved amino acid not in a metal-binding motif. H869A is well expressed with a normal kcat. However, the Km for cAMP and the IC50 for cGMP are each fourfold greater in the mutant than those in the wild-type recombinant, suggesting that this histidine is in the inhibitory binding site. Three well-conserved cysteines were mutated, but C942A, C945A, and C1013A all had kinetic values similar to the wild type. The results have identified a histidine at the active site essential for catalysis and a second histidine important for inhibitor binding and have further supported the essential nature of the unique 44-amino acid insert.
磷酸二酯酶（PDE）通过将环磷酸腺苷（cAMP）水解为5'-AMP，对细胞内第二信使cAMP水平的下调起重要作用。我们实验室之前的研究表明，人血小板PDE3A活性位点有两个必需的组氨酸残基和一个半胱氨酸残基。因此，我们决定开始对PDE3A保守结构域中的组氨酸和半胱氨酸进行突变。一种截短但具有催化活性的重组PDE3A蛋白在酵母细胞中表达。在羧基末端设计了一个六个组氨酸的标签，以便通过镍柱进行蛋白质纯化。这个cDNA构建体PDE3ADelta1用于构建突变体。在PCR或DNA复制过程中，通过突变寡核苷酸引入突变，并通过测序进行确认。突变的cDNA在缺乏PDE的酵母宿主酿酒酵母菌株GL62中表达。使用兔抗血小板PDE3A多克隆抗体通过蛋白质印迹法监测重组PDE突变蛋白的表达水平。测定每个突变体对环磷酸鸟苷（cGMP）的kcat、KM和IC50以及对米力农的IC50。两个高度保守的组氨酸和四个保守的半胱氨酸分别突变为丙氨酸。C816存在于PDE3特有的44个氨基酸插入序列中。突变体C816A表达不佳（>2%），可能折叠不当。H840是金属结合基序HDXXH中第二个基序的第二个组氨酸。突变体H840A虽然表达适度良好，但活性检测不到，可能负责结合催化所需的二价阳离子，例如Mn2+。H869是一个高度保守的氨基酸，不在金属结合基序中。H869A表达良好，kcat正常。然而，突变体中cAMP的Km和cGMP的IC50分别比野生型重组体大四倍，表明这个组氨酸位于抑制性结合位点。三个保守的半胱氨酸被突变，但C942A、C945A和C1013A的动力学值都与野生型相似。这些结果确定了活性位点上一个对催化至关重要的组氨酸和另一个对抑制剂结合重要的组氨酸，并进一步支持了独特的44个氨基酸插入序列的本质重要性。
