Partial characterization of the active site human platelet cAMP phosphodiesterase, PDE3A, by site-directed mutagenesis.
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.
Cheung
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Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, Pennsylvania, 19140, USA.
Yu
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Zhang
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Colman
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D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001792:Blood Platelets / Q000201:enzymology*; D002384:Catalysis; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D003545:Cysteine / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D007700:Kinetics; D020105:Milrinone / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D006639:Histidine; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D006152:Cyclic GMP; D020105:Milrinone; D003545:Cysteine