Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5.

Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cCMP-specific PDE (PDES), we showed previously that point mutation of nine of these profoundly decreases k(cat) when the assay is conducted in the presence of Mg2+; seven of these are in the prototypical metal-binding motifs A and B (HX3HXnE) that we identified earlier. Tandem arrangement of two of these metal-binding motifs in PDEs is novel, and whether residues within these motifs are involved in metal support of catalytic activity is a fundamental question in this field. This report shows that mutation of either His-607 (A motif) or His-643 (B motif) to alanine profoundly diminishes support of PDE catalysis by Mn2+ or Mg2+, but mutation of His-647 in B motif or of Glu in either motif does not. H607A and H643A mutants have much greater maximum catalytic rates supported by Mn2+ than that by Mg2+; catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A, K(a)s for Mn2+ and Mg2+ are increased, but the effect of Mn2+ is 2-fold greater than that of Mg2+ in each. Mutation of any of the other conserved residues (Asn-604, Asp-644, His-675, Asp-714, and Asp-754) causes unremarkable changes in Mn2+ or Mg2+ support Of catalysis. This study identifies specific residues in PDES that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX3HXnE motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.