Identification of key amino acids in a conserved cGMP-binding site of cGMP-binding phosphodiesterases. A putative NKXnD motif for cGMP binding.
J Biol Chem
; 271(36): 22240-4, 1996 Sep 06.
Article
en En
| MEDLINE
| ID: mdl-8703039
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.
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Banco de datos:
MEDLINE
Asunto principal:
3',5'-GMP Cíclico Fosfodiesterasas
Tipo de estudio:
Diagnostic_studies
Límite:
Animals
Idioma:
En
Año:
1996
Tipo del documento:
Article