Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5)

The known mammalian 3':5'-cyclic nucleotide phosphodiesterases (PDEs) contain a conserved region located toward the carboxyl terminus, which constitutes a catalytic domain, To identify amino acids that are important for catalysis, we introduced substitutions at 23 conserved residues within the catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (cGBPDE; PDES). Wild-type and mutant proteins were compared with respect to K-m for cGMP, k(cat) and IC50 for zaprinast, The most dramatic decrease in k(cat) was seen with H643A and D754A mutants with the decrease in free energy of binding (Delta Delta G(T)) being about 4.5 kcal/mol for each, which is within the range predicted for loss of a hydrogen bond involving a charged residue, His(643)?S and Asp(754) conserved in all known PDEs and are sarong candidates to be directly involved in catalysis, Substitutions of His(603), His(607), His(647), Glu(672), Asp(714) also produced marked changes in k(cat) and these residues are likely to be important for efficient catalysis, The Y602A and E775A mutants exhibited the most dramatic increases in K-m for cGAMP, with calculated Delta Delta G(T) of 2.9 and 2.8 kcaj/mol, respectively, that these two residues are important for cGMP binding in the catalytic site, Zaprinast is a potent competitive inhibitor of cGB-PDE, but the key residues for its binding differ significantly from those that bind cGMP.