Kinetic and Structural Studies of Phosphodiesterase-8A and Implication on the Inhibitor Selectivity

• Published in: Biochemistry (Easton) Vol. 47; no. 48; pp. 12760 - 12768
• Main Authors: Wang, Huanchen, Yan, Zier, Yang, Serena, Cai, Jiwen, Robinson, Howard, Ke, Hengming
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 02.12.2008
• Subjects: 1-Methyl-3-isobutylxanthine - metabolism; 1-Methyl-3-isobutylxanthine - pharmacology; 3',5'-Cyclic-AMP Phosphodiesterases - antagonists & inhibitors; 3',5'-Cyclic-AMP Phosphodiesterases - chemistry; 3',5'-Cyclic-AMP Phosphodiesterases - genetics; 3',5'-Cyclic-AMP Phosphodiesterases - metabolism; Amino Acid Sequence; Catalytic Domain; Crystallography, X-Ray; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Escherichia coli - genetics; Humans; Kinetics; Models, Molecular; Molecular Sequence Data; Mutation; Protein Renaturation; Substrate Specificity; Tyrosine
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi801487x

Cyclic nucleotide phosphodiesterase-8 (PDE8) is a family of cAMP-specific enzymes and plays important roles in many biological processes, including T-cell activation, testosterone production, adrenocortical hyperplasia, and thyroid function. However, no PDE8 selective inhibitors are available for trial treatment of human diseases. Here we report kinetic properties of the highly active PDE8A1 catalytic domain prepared from refolding and its crystal structures in the unliganded and 3-isobutyl-1-methylxanthine (IBMX) bound forms at 1.9 and 2.1 Å resolutions, respectively. The PDE8A1 catalytic domain has a K M of 1.8 μM, V max of 6.1 μmol/min/mg, a k cat of 4.0 s−1 for cAMP, and a K M of 1.6 mM, V max of 2.5 μmol/min/mg, a k cat of 1.6 s−1 for cGMP, thus indicating that the substrate specificity of PDE8 is dominated by K M. The structure of the PDE8A1 catalytic domain has similar topology as those of other PDE families but contains two extra helices around Asn685-Thr710. Since this fragment is distant from the active site of the enzyme, its impact on the catalysis is unclear. The PDE8A1 catalytic domain is insensitive to the IBMX inhibition (IC50 = 700 μM). The unfavorable interaction of IBMX in the PDE8A1−IBMX structure suggests an important role of Tyr748 in the inhibitor binding. Indeed, the mutation of Tyr748 to phenylalanine increases the PDE8A1 sensitivity to several nonselective or family selective PDE inhibitors. Thus, the structural and mutagenesis studies provide not only insight into the enzymatic properties but also guidelines for design of PDE8 selective inhibitors.