PMID: 9187266Jun 1, 1997Paper

Role of conserved histidines in catalytic activity and inhibitor binding of human recombinant phosphodiesterase 4A

Molecular Pharmacology
S JacobitzT J Torphy

Abstract

To identify critical amino acids within the central conserved region of recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE4A), we engineered the expression of point mutants in a fully active rhPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were changed independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to bind prototypical PDE4 inhibitors [3H]-(R)-rolipram or [3H]RP 73401 was also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests that these changes resulted from non-localized perturbations in the structure of the enzyme. More interesting results were obtained when histidine residues at positions 505 and 506 were changed independently to aspar agines. The K(m) value for cAMP increased 3-fold in H505N (K(m) = 11 +/- 3 microM) and 11-fold in H506N (K(m) = 44 +/- 6 microM) compared with the wild-type protein (K(m) = 4 +/- 1 microM). These mutant proteins bound [3H]-(R)-rolipram and [3H]RP 73401 with K(d) values of 1.8 +/- 0.4 and 0.3 +/- 0.1 nM, respectively, for H505N, and ...Continue Reading

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Citations

Feb 13, 1999·The Journal of Biological Chemistry·J M AtienzaJ Colicelli
Dec 26, 2001·Molecular Pharmacology·Orly DymJohn Colicelli
Nov 23, 2005·Expert Opinion on Therapeutic Targets·Kam Y J ZhangGideon Bollag
Feb 12, 1998·Biochemical and Biophysical Research Communications·M D PercivalJ P Falgueyret
Nov 10, 2000·Brain Research. Molecular Brain Research·B E ThompsonJ A Cherry

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