Zinc interactions and conserved motifs of the cGMP-binding cGMP-specific phosphodiesterase suggest that it is a zinc hydrolase.
cGMP-binding cGMP-specific phosphodiesterase (cG-BPDE) binds tightly to a Zn(2+)-chelate column (Francis, S. H., and Corbin, J. D. (1988) Methods Enzymol. 159, 722-729). Using three different approaches, Zn2+ is now shown to bind to cG-BPDE, and the Kd is determined to be approximately 0.5 microM, with a binding stoichiometry of approximately 3 mol of Zn2+/mol of monomer. A similar concentration range of Zn2+ (0.05-1 microM Zn2+) also supports phosphodiesterase (PDE) catalytic activity. The Zn2+ binding to cG-BPDE is not diminished by, nor is catalysis supported by, relatively high concentrations of Cu2+, Cd2+, Ca2+, or Fe2+. Neither cGMP nor 3-isobutyl-1-methylxanthine affects Zn2+ binding under the conditions used. Mn2+, Co2+, or Mg2+ supports catalysis, but only at significantly higher concentrations (4-, 15-, and 250-fold, respectively) than that required for Zn2+. Two tandem amino acid sequences, which are conserved in the catalytic domains of all characterized mammalian PDEs, resemble the single sequence motif that has been shown to coordinate Zn2+ in the catalytic sites of Zn2+ hydrolases such as thermolysin.
Francis
S H
SH
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0615.
Colbran
J L
JL
McAllister-Lucas
L M
LM
Corbin
J D
JD
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002413:Cations, Divalent / Q000494:pharmacology; D017124:Conserved Sequence; D006152:Cyclic GMP / Q000378:metabolism*; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000378:metabolism*
D002413:Cations, Divalent; D008670:Metals; D006867:Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP; D015032:Zinc