An essential aspartic acid at each of two allosteric cGMP-binding sites of a cGMP-specific phosphodiesterase.
The amino acid sequences of all known cGMP-binding phosphodiesterases (PDEs) contain internally homologous repeats (a and b) that are 80-90 residues in length and are arranged in tandem within the putative cGMP-binding domains. In the bovine lung cGMP-binding, cGMP-specific PDE (cGB-PDE or PDE5A), these repeats span residues 228-311 (a) and 410-500 (b). An aspartic acid (residue 289 or 478) that is invariant in repeats a and b of all known cGMP-binding PDEs was changed to alanine by site-directed mutagenesis of cGB-PDE, and wild type (WT) and mutant cGB-PDEs were expressed in COS-7 cells. Purified bovine lung cGB-PDE (native) and WT cGB-PDE displayed identical cGMP-binding kinetics, with approximately 1.8 microM cGMP required for half-maximal saturation. The D289A mutant showed decreased affinity for cGMP (Kd > 10 microM) and the D478A mutant showed increased affinity for cGMP (Kd approximately 0.5 microM) as compared to WT and native cGB-PDE. WT and native cGB-PDE displayed an identical curvilinear profile of cGMP dissociation which was consistent with the presence of distinct slowly dissociating (koff = 0.26 h-1) and rapidly dissociating (koff = 1.00 h-1) sites of cGMP binding. In contrast, the D289A mutant displayed a single koff = 1.24 h-1, which was similar to the calculated koff for the fast site of WT and native cGB-PDE, and the D478A mutant displayed a single koff = 0.29 h-1, which was similar to that calculated for the slow site of WT and native cGB-PDE. These results were consistent with the loss of a slow cGMP-binding site in repeat a of the D289A mutant cGB-PDE, and the loss of a fast site in repeat b of the D478A mutant, suggesting that cGB-PDE possesses two distinct cGMP-binding sites located at repeats a and b, with the invariant aspartic acid being crucial for interaction with cGMP at each site.
McAllister-Lucas
L M
LM
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0165, USA.
Haik
T L
TL
Colbran
J L
JL
Sonnenburg
W K
WK
Seger
D
D
Turko
I V
IV
Beavo
J A
JA
Francis
S H
SH
Corbin
J D
JD
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D001483:Base Sequence; D015153:Blotting, Western; D002417:Cattle; D006152:Cyclic GMP / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D001224:Aspartic Acid; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP