Histidine-607 and Histidine-643 Provide Important Interactions for Metal Support of Catalysis in Phosphodiesterase-5

Abstract

Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cGMP-specific PDE (PDE5), we showed previously that point mutation of nine of these profoundly decreases k(cat) when the assay is conducted in the presence of Mg(2+); seven of these are in the prototypical metal-binding motifs A and B (HX(3)HX(n)()E) 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 Mn(2+) or Mg(2+), 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 Mn(2+) than that by Mg(2+); catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A, K(a)s for Mn(2+) and Mg(2+) are increased, but the effect of Mn(2+) is 2-fold greater than that of Mg(2+) 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 Mn(2+) or Mg(2+) support of catalysis. This study identifies specific residues in PDE5 that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX(3)HX(n)()E motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.

Crystal Structures of Phosphodiesterases 4 and 5 in Complex with Inhibitor 3-Isobutyl-1-methylxanthine Suggest a Conformation Determinant of Inhibitor Selectivity

“…These six coordinations form an octahedron and are the same as in PDE4 (31,33). The role of the equivalent residues His-607 and His-643 of bovine PDE5 in metal coordination was established by site-directed mutagenesis (42). The second metal ion forms an octahedron with Asp-654 and five bound water molecules and has the same coordinations as in PDE4D2.…”

Identification of the γ Subunit-interacting Residues on Photoreceptor cGMP Phosphodiesterase, PDE6α′

“…The two metal atoms, apparently a tightly bound Zn 2ϩ and a more loosely associated Mg 2ϩ , are central to the hydrolysis of cyclic nucleotides by PDE6 (37). Corresponding residues, His 607 and His 643 , are necessary for the metal support of catalysis in PDE5 (38). Another important residue within the PDE6␣Ј catalytic pocket is conserved Gln 771 (Fig.…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…A unique metal-binding site in PDEs that utilizes two metal ions with one of them being zinc was first suggested in studies with PDE5 (109,116). However, a role for zinc in PDEs was not widely accepted until publication of the first x-ray crystallographic structure of a PDE4 C domain (417), which revealed a novel binuclear metal-ion binding site in which zinc was tightly bound in one site, i.e., the Me-1 site.…”