Probing domain functions of chimeric PDE6alpha'/PDE5 cGMP-phosphodiesterase.
Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6alpha') in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6alpha'/PDE5 enzyme containing the PDE6alpha' noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6alpha'. The inhibitory gamma subunit of PDE6 (Pgamma) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6alpha' by approximately 6-fold. The polycationic region of Pgamma, Pgamma-24-45, was mainly responsible for this effect, while the inhibitory domain of Pgamma, Pgamma-63-87, was ineffective. On the contrary, Pgamma failed to inhibit catalytic activity of the chimeric PDE6alpha'/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6alpha' are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn402 --> Ala resulted in an approximately 10-fold increase of the Km value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6alpha' may regulate catalytic properties of the enzyme.
Granovsky
A E
AE
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Natochin
M
M
McEntaffer
R L
RL
Haik
T L
TL
Francis
S H
SH
Corbin
J D
JD
Artemyev
N O
NO
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000378:metabolism; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology*; D012374:Rod Cell Outer Segment / Q000201:enzymology*; D018411:Spodoptera; D014162:Transfection
D011993:Recombinant Fusion Proteins; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006152:Cyclic GMP; D000409:Alanine