Vol. 57, Issue 5, 991-999, May 2000

Analysis of a Mutation in Phosphodiesterase Type 4 that Alters Both Inhibitor Activity and Nucleotide Selectivity

Sarah B. Herman, Dawn M. Juilfs, Eric B. Fauman, Paul Juneau, and Joseph P. Menetski

Departments of Molecular Biology (S.B.H., D.M.J., J.P.M.), Biomolecular Structure and Drug Design (E.B.F.), and Nonclinical Biometrics (P.J.), Parke-Davis Pharmaceutical Research/Division of Warner-Lambert, Ann Arbor, Michigan

Cyclic nucleotide phosphodiesterase type 4 (PDE4) is a cAMP-specific phosphodiesterase that is found as four distinct genes in the mammalian genome (PDE4A, 4B, 4C, and 4D). Mutation analysis was done to identify the amino acids involved in activity and inhibitor selectivity. Mutations at Asp333 were made in HSPDE4D3 based on mutations that affect rolipram sensitivity in RNPDE4B1. The PDE4D3 Asp-Asn mutant was resistant to inhibition by rolipram as well as several other PDE4 inhibitors tested. These results suggest that this residue is near the inhibitor binding pocket in PDE4D3. Sequence comparison of PDE4 with cGMP-specific PDE proteins shows a conserved aspartic acid at position 333 in PDE4D3 and a conserved asparagine at this position in PDE enzymes that hydrolyze cGMP. Therefore, cGMP hydrolysis by PDE4D3 Asp-Asn was measured. PDE4D3 Asp-Asn hydrolyzes cGMP with kinetic constants similar to those observed for this protein with cAMP (K_m ~ 20 µM, V_max ~ 2 µmol AMP/min/mg recombinant protein). Under identical conditions, the K_m value for cAMP hydrolysis by wild-type PDE4D3 is 3 µM and the V_max value is 1 µmol AMP/min/mg recombinant protein. In addition, the PDE4D3 Asp-Ala mutant protein could hydrolyze cGMP. Finally, the analogous mutation in HSPDE4B1 (Asp413Asn) also allows hydrolysis of cGMP. These results show that this aspartic acid residue is important in inhibitor binding and nucleotide discrimination and suggest this residue is in the active site of PDE4.