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 nonlocalized 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. TheK _m value for cAMP increased 3-fold in H505N (K _m = 11 ± 3 μm) and 11-fold in H506N (K _m = 44 ± 6 μm) compared with the wild-type protein (K _m = 4 ± 1 μm). 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 3.9 ± 0.9 and 0.5 ± 0.1 nm, respectively, for H506N. These values are nearly identical to those obtained with the wild-type rhPDE4A/Met201–886. In contrast, the IC₅₀ values for cAMP competition with either [³H]-(R)-rolipram or [³H]RP 73401 binding increased ∼2-fold in H505N and ∼13-fold in H506N compared with the wild type protein. These increases are virtually identical to the changes in theK _m value for cAMP in these mutants. We conclude that His506 and, perhaps, His505 are involved in binding of cAMP to PDE4A/Met201–886 but not in binding of PDE4-selective inhibitors.