ROLE OF CONSERVED HISTIDINES IN CATALYTIC ACTIVITY AND INHIBITOR BINDING OF HUMAN RECOMBINANT PHOSPHODIESTERASE 4A

To identify critical amino acids within the central conserved region o f recombinant human cAMP-specific phosphodiesterase 4 subtype A (rhPDE 4A), we engineered the expression of point mutants in a fully active r hPDE4A/Met201-886. When histidine residues at positions 433, 437, 473, and 477, which are highly conserved among all PDE families, were chan ged independently to serine residues, cAMP hydrolyzing activities were substantially reduced or abolished. The ability of these mutants to b ind prototypical PDE4 inhibitors [H-3]-(R)-rolipram or [H-3]RP 73401 w as also decreased in parallel with the loss of catalytic activity. The parallel loss of catalytic activity and inhibitor binding suggests th at these changes resulted from nonlocalized perturbations in the struc ture of the enzyme. More interesting results were obtained when histid ine residues at positions 505 and 506 were changed independently to as paragines. The K-m value for cAMP increased 3-fold in H505N (K-m = 11 +/- 3 mu M) and 11-fold in H506N (K-m = 44 +/- 6 mu M) compared with t he wild-type protein (K-m = 4 +/- 1 mu M). These mutant proteins bound [H-3]-(R)-rolipram and [H-3]RP 73401 with K-d values of 1.8 +/- 0.4 a nd 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 t o those obtained with the wild-type rhPDE4A/Met201-886. In contrast, t he IC50 values for cAMP competition with either [H-3]-(R)-rolipram or [H-3]RP 73401 binding increased similar to 2-fold in H505N and similar to 13-fold in H506N compared with the wild type protein. These increa ses are virtually identical to the changes in the K-m value for cAMP i n these mutants. We conclude that His506 and, perhaps, His505 are invo lved in binding of cAMP to PDE4A/Met201-886 but not in binding of PDE4 -selective inhibitors.