Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase.
The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain. To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGB-PDE; PDE5). Wild-type and mutant proteins were compared with respect to Km for cGMP, kcat, and IC50 for zaprinast. The most dramatic decrease in kcat was seen with H643A and D754A mutants with the decrease in free energy of binding (DeltaDeltaGT) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue. His643 and Asp754 are conserved in all known PDEs and are strong candidates to be directly involved in catalysis. Substitutions of His603, His607, His647, Glu672, and Asp714 also produced marked changes in kcat, and these residues are likely to be important for efficient catalysis. The Y602A and E775A mutants exhibited the most dramatic increases in Km for cGMP, with calculated DeltaDeltaGT of 2.9 and 2.8 kcal/mol, respectively, that these two residues are important for cGMP binding in the catalytic site. Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.
Turko
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Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
Francis
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Corbin
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D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011688:Purinones / Q000494:pharmacology; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013816:Thermodynamics
D010726:Phosphodiesterase Inhibitors; D011688:Purinones; D011994:Recombinant Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C011145:zaprinast; D006152:Cyclic GMP