title: Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding phosphodiesterases. A putative NKXnD motif for cGMP binding.
abstract: cGMP-binding phosphodiesterases contain two kinetically distinct cGMP-binding sites (a and b), and each site contains a conserved N(K/R)XnFX3DE sequence. N276A, K277A, K277R, D289A, and E290A mutants in the N276KX7FX3DE290 sequence of site a (higher affinity site) of bovine cGMP-binding, cGMP-specific phosphodiesterase (cGB-PDE or PDE5A) were expressed in High Five cells and purified. The cGMP-binding affinities of three mutants [K277A (Kd approximately 12 microM), D289A (Kd approximately 24 microM), and N276A (Kd approximately 60 microM)] were decreased in comparison with wild-type enzyme (Kd = 1.3 microM), which suggested an important role for Asn276, Lys277, and Asp289 in cGMP binding. These residues could be presented as a putative NKXnD motif, and their functions were predicted based on analogy with the canonical NKXD motif in GTP-binding proteins. No marked differences in catalytic functions such as specific activity, Km for cGMP, and IC50 for zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does not influence the catalytic properties of cGB-PDE.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA.
authors.forename: I V
authors.initials: IV
authors.lastname: Turko
authors.forename: T L
authors.initials: TL
authors.lastname: Haik
authors.forename: L M
authors.initials: LM
authors.lastname: McAllister-Lucas
authors.forename: F
authors.initials: F
authors.lastname: Burns
authors.forename: S H
authors.initials: SH
authors.lastname: Francis
authors.forename: J D
authors.initials: JD
authors.lastname: Corbin
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data
chemical_list: D000596:Amino Acids; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP