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Analysis of a mutation in phosphodiesterase type 4 that alters both inhibitor activity and nucleotide selectivity
S B Herman1, D M Juilfs, E B Fauman
1Department of Molecular Biology, Parke-Davis Pharmaceutical Research/Division of Warner-Lambert, Ann Arbor, MI 48105, USA.
Abstract:
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) approximately 20 microM, V(max) approximately 2 micromol AMP/min/mg recombinant protein). Under identical conditions, the K(m) value for cAMP hydrolysis by wild-type PDE4D3 is 3 microM and the V(max) value is 1 micromol 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.
Insights
Mutation analysis of cyclic nucleotide phosphodiesterase type 4 (PDE4) identified Asp333 as crucial for inhibitor binding and nucleotide discrimination. This residue
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Cyclic nucleotide phosphodiesterase type 4 (PDE4) enzymes regulate intracellular cAMP levels.
- PDE4 enzymes are targets for various therapeutic applications.
- Understanding PDE4 structure and function is key to developing selective inhibitors.
Purpose of the Study:
- To identify amino acid residues critical for PDE4 activity and inhibitor selectivity.
- To investigate the role of Asp333 in the PDE4D3 active site.
- To explore the impact of mutations on PDE4 substrate specificity.
Main Methods:
- Site-directed mutagenesis of human PDE4D3 at Asp333.
- Enzyme kinetics assays for cAMP and cGMP hydrolysis.
- Inhibition assays using rolipram and other PDE4 inhibitors.
- Sequence comparison between PDE4 and cGMP-specific PDE enzymes.
Main Results:
- Mutation of Asp333 to Asparagine (Asp-Asn) in PDE4D3 conferred resistance to rolipram and other PDE4 inhibitors.
- The PDE4D3 Asp-Asn mutant exhibited significant cGMP hydrolytic activity, similar to its cAMP hydrolysis kinetics.
- Mutations at Asp333 in both PDE4D3 and PDE4B1 enabled cGMP hydrolysis, indicating a role in nucleotide discrimination.
- The Asp-Asn mutation suggests Asp333 is located near the inhibitor binding pocket and within the active site.
Conclusions:
- The aspartic acid residue at position 333 is critical for inhibitor binding and substrate selectivity in PDE4 enzymes.
- This residue plays a key role in distinguishing between cAMP and cGMP hydrolysis.
- The findings provide insights into the active site architecture of PDE4 and guide the design of selective inhibitors.