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 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.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615, USA. sharron.francis@mcmail.vanderbilt.edu
S H
SH
Francis
I V
IV
Turko
K A
KA
Grimes
J D
JD
Corbin
D015106:3',5'-Cyclic-GMP Phosphodiesterases; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D002417:Cattle; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D006639:Histidine / Q000737:chemistry* / Q000235:genetics; D008274:Magnesium / Q000737:chemistry; D008345:Manganese / Q000737:chemistry; D008670:Metals / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation
D002413:Cations, Divalent; D008670:Metals; D008345:Manganese; D006639:Histidine; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D008274:Magnesium