Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5.
Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.
Granovsky
A E
AE
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Artemyev
N O
NO
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003001:Cloning, Molecular; D003198:Computer Simulation; D017124:Conserved Sequence; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020419:Photoreceptor Cells, Vertebrate / Q000201:enzymology*; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012984:Software; D014714:Vertebrates
D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D054707:Cyclic Nucleotide Phosphodiesterases, Type 6; D000409:Alanine