Mutational mapping of kinetic and pharmacological properties of a human cardiac cAMP phosphodiesterase.
We have created a series of deletion mutants of a human cardiac cAMP phosphodiesterase in order to define sequences necessary for function and to identify residues required for inhibition by cGMP and by the drugs milrinone and trequinsin. These truncated constructs were expressed in yeast cells, and their biochemical properties were analyzed. The mutations define an amino acid sequence that is essential for function. Among the active constructs, there was considerable variability in the level of expression and in the stability of the proteins, with the full-length and near full-length constructs being the least stable. There were, however, no significant changes in Km values among the active enzymes. Cation studies confirmed that Mn2+ is a more efficient cofactor than Mg2+ or Co2+. Interestingly, Mn2+ acts as a more efficient cofactor for cGMP inhibition as well. Although IC50 values for the drugs trequinsin and milrinone were not significantly altered by deletions, there was a decrease in cGMP IC50 values for the smaller constructs, indicating a role for amino acid residues outside the catalytic region in cGMP inhibition. We also demonstrate in vivo inhibition of this enzyme in yeast cells grown in the presence of pharmacological inhibitors, allowing for the selection of drug-resistant mutants. Finally, we have constructed and analyzed chimeric genes in which portions of this phosphodiesterase are replaced with homologous sequences from a closely related phosphodiesterase isozyme that is expressed in brain. Our results demonstrate that sequence variations between related isozymes account for more than just pharmacological distinctions and may reflect significant structural differences.
Pillai
R
R
Department of Biological Chemistry, UCLA School of Medicine 90024.
Staub
S F
SF
Colicelli
J
J
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D006152:Cyclic GMP / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D017931:DNA Primers; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology*; D007700:Kinetics; D020105:Milrinone; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011728:Pyridones / Q000494:pharmacology*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D017384:Sequence Deletion; D044005:Tetrahydroisoquinolines
D017931:DNA Primers; D007546:Isoquinolines; D010726:Phosphodiesterase Inhibitors; D011728:Pyridones; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D044005:Tetrahydroisoquinolines; C035964:trequinsin; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D020105:Milrinone