IDENTIFICATION OF KEY AMINO-ACIDS IN A CONSERVED CGMP-BINDING SITE OFCGMP-BINDING PHOSPHODIESTERASES - A PUTATIVE NKX(N)D MOTIF FOR CGMP BINDING

cGMP-binding phosphodiesterases contain two kinetically distinct cGMP- binding sites (a and b), and each site contains a conserved N(K/R)X(n) FX(3)DE sequence, N276A, K277A, R277R, D289A, and E290A mutants in the N(276)KX(7)FX(3)DE(290) 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 aff inities of three mutants [K277A (K-d approximate to 12 mu M), D289A (K -d approximate to 24 mu M) and N276A (K-d approximate to 60 mu M)] wer e decreased in comparison with wild-type enzyme (K-d = 1.3 mu M), whic h suggested an important role for Asn(276), Lys(277), and Asp(289) in cGMP binding, These residues could be presented as a putative NKX(n)D motif, and their functions were predicted based on analogy with the ca nonical NKXD motifin GTP-binding proteins, No marked differences in ca talytic functions such as specific activity, K-m for cGMP, and IC50 fo r zaprinast or 3-isobutyl-1-methylxanthine were found among wild-type and mutant cGB-PDEs. This suggested that cGMP binding to site a does n ot influence the catalytic properties of cGB-PDE.