Critical role of conserved histidine pairs HNXXH and HDXXH in recombinant human phosphodiesterase 4A.

Cyclic AMP-Phosphodiesterases (cAMP-PDEs) catalyse the hydrolysis cAMP to AMP and thus serve to modulate the ligand-->adenylate cyclase-->cAMP-->PKA signal transduction pathway. PDEs exist as a multigene family of enzymes that bear significant sequence homology in the catalytic domains. The sequence alignment of these domains has revealed the presence of two histidine motifs: motif I, HNXXH, and motif II, HDXXH. These amino acid sequences are canonical motifs, which act as ligands for divalent metal cations required for catalytic activity. In this paper, we report human monocyte PDE4A to be a zinc-binding protein. Substitution by site-directed mutagenesis of either histidine in motif I by serine, which is not a ligand for metals, results in complete loss of catalytic activity and loss of sensitivity to divalent metal cation activation. However, similar mutations in motif II gave proteins that retained both approximately 50% of initial activity and the ability to respond differentially to Mg2+, Mn2+ and Co2+. Moreover the motif II mutants exhibited both functional group requirements and retained their pKa values. When the inactive mutants were affinity-labelled with 8-BDB-TcAMP and probed with antibody against cAMP or antibody against PDE4A, Western blots were unaltered. These results show that the conserved histidines in motif I are an absolute requirement for catalytic activity, whereas motif II histidines are required only to achieve maximum activity.

Cyclic AMP-specific phosphodiesterase inhibitor rolipram and RO-20-1724 promoted apoptosis in HL60 promyelocytic leukemic cells via cyclic AMP-independent mechanism.

Phosphodiesterases (PDEs) are responsible for the hydrolysis of cAMP and cGMP which act as intracellular second messengers in a variety of cellular functions. In this paper we report that PDE3 and PDE4 were two dominant classes of PDEs expressed in HL60 cells. The influence of specific PDE inhibitors on apoptosis in HL60 cells was studied. The non-specific inhibitor IBMX and PDE3 specific inhibitors (milrinone and trequinsin) did not promote apoptosis. They inhibited apoptosis induced by paclitaxel or thapsigargin. However, PDE4 specific inhibitors (rolipram and RO-20-1724) promoted apoptosis within 5 h. In HL60 cells, other cAMP-eliciting reagents (8-bromo-cAMP, Sp-cAMP and forskolin) also inhibited apoptosis, while cell-permeable cGMP analogs did not affect apoptosis. Therefore, IBMX and PDE3 specific inhibitors may prevent HL60 cells from apoptosis by increasing intracellular cAMP. However, apoptosis induced by PDE4 specific inhibitors is not likely due to increased cAMP level. These results suggest that rolipram and RO-20-1724 promoted apoptosis in HL60 cells through cAMP-independent mechanism.

Inactivation of recombinant monocyte cAMP-specific phosphodiesterase by cAMP analog, 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate.

Two cAMP analogs, 8- and 2- [(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8- and 2-BDB-TcAMP) have been used in probing the catalytic site of recombinant monocyte cAMP-specific phosphodiesterase (PDE4a). 2-BDB-TcAMP is a reversible and competitive inhibitor (Ki = 5.5 mumol/L) of cAMP hydrolysis by PDE4a, 8-BDB-TcAMP irreversibly inactivates the enzyme in a time- and concentration-dependent manner with a second order rate constant of 0.022 mmol/L-1 min-1. The rate of inactivation of PDE4a is reduced by the presence of the substrate cAMP and specific inhibitors, rolipram and denbufylline, but not by cGMP or AMP. Reduction of the enzyme-inhibitor complex with sodium [3H]borohydride shows that 1.2 mol of the affinity label/mol of enzyme was incorporated. The radiolabeled peptide is composed of 10 amino acid residues (697 to 706) located near the carboxyl end of the proposed catalytic domain. The peptide (GPGHPPLPDK) has seven nonpolar and aliphatic residues, of which four are proline, giving the peptide a highly structured conformation. This peptide is the first to be identified in the putative catalytic domain involved in substrate recognition.

Evidence for the presence of essential histidine and cysteine residues in platelet cGMP-inhibited phosphodiesterase.

Camp is a major regulator of platelet function. cGMP-inhibited phosphodiesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cAMP. The pH-rate profile plot for this enzyme yields pKa values of 6.5 and 9.0, consistent with histidine and cysteine residues respectively. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and concentration-dependent manner, and this effect was rapidly reversed by hydroxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as deduced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzyme activity. N-Ethylmaleimide (NEM) and 5.5'-dithiobis-(2-nitrobenzoic acid) (DTNB) inactivate cGI-PDE in a time- and concentration-dependent manner, suggesting the selective modification of a cysteine residue. AMP protects the enzyme against DEP, NEM and DTNB, suggesting the presence of histidine and cysteine residues at the active site of cGI-PDE. [14C]DEP incorporation in the presence of AMP or cGMP indicates the protection of two histidine residues by each nucleotide. These residues are different for each agent, since the combination of AMP and cGMP protects four histidine residues. [3H]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PDE was protected by AMP, but not only by cGMP. We conclude that cGI-PDE possesses two essential histidine residues for activity, two additional histidines for cGMP inhibition, and one cysteine residue at the active site.

Divalent metal cation requirement and possible classification of cGMP-inhibited phosphodiesterase as a metallohydrolase.

cGMP-inhibited phosphodiesterase (cGI-PDE) has been found to require a divalent metal cation for cAMP hydrolysis. The cGI-PDE isolated from human platelets exhibited significantly higher enzymatic activity when incubated with Mn2+, Mg2+, and Co2+. The addition of Zn2+, Cd2+, Ca2+, K+, or Na+ to the enzyme did not enhance the activity and, when present in high concentration (> 1.0 microM), Zn2+ and Cd2+ inhibited the enzymatic activity of cGI-PDE. The inhibition by Zn2+ (and Cd2+) was partially prevented by preincubation of the enzyme with Mn2+. The enzyme was also inhibited by metal chelators EDTA and 1,10-phenanthroline and not by their non-metal-chelating analogs. The partial protection against chelation (and inhibition) was afforded by AMP (the product of cAMP hydrolysis).

Utilization of copper as a paramagnetic probe for the binuclear metal center of phosphotriesterase.

Bacterial phosphotriesterase catalyzes the hydrolysis of organophosphate triesters. To be active, the enzyme requires that two divalent cations are bound. These metal ions are bound in close proximity to one another as a binuclear center. To characterize the structure and function of the binuclear metal binding sites, we have prepared the copper-substituted enzyme. The kinetic data indicate that this enzyme is essentially inactive toward the hydrolysis of phosphotriesters. The EPR signal arising from the copper-substituted enzyme is nearly axial, with g parallel = 2.24 and g perpendicular = 2.05 and shows at least seven superhyperfine transitions in the g perpendicular region with A perpendicular = 1.45 x 10(-3) cm-1. These splittings are consistent with the direct ligation of more than one nitrogen to the metal center. The average spin quantitation of copper-substituted enzymes are 0.6 spin/Cu, approximately half of that observed for noninteracting Cu2+ ions. The spin intensity increases to ca. 1 spin/Cu when samples are denatured with acid. The binding of metal ions to the designated alpha and beta sites is highly synergistic (i.e., the metal ions bind in pairs). Mixed metal complexes of the type Cu/X and X/Cu were prepared. When X is a diamagnetic ion (Zn2+ or Cd2+), the spin quantitation increases, but when X is the paramagnetic Co2+ ion, the spin quantitation decreases. This behavior indicates that the low spin intensities observed for copper-substituted phosphotriesterase arise from spin-coupling of the two adjacent Cu2+ ions. The addition of dithiothreitol, ascorbate, or dithionite to the copper-substituted phosphotriesterase results in nearly the complete loss of spin intensity. This indicates that the bound coppers can be reduced to the cuprous state.

Structural characterization of the divalent cation sites of bacterial phosphotriesterase by 113Cd NMR spectroscopy.

The phosphotriesterase from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters. The isolated native protein contains zinc, and removal of this metal abolishes the enzymatic activity. Reconstitution of the apoenzyme requires 2 mol of cadmium per mol of protein for full catalytic activity. The kcat and Km values for the hydrolysis of paraoxon for the cadmium-substituted enzyme are 4300 s-1 and 390 microM, respectively. These values compare favorably with the kinetic constants observed for the zinc-substituted enzyme (2300 s-1 and 78 microM). A hybrid enzyme containing one zinc and one cadmium ion is catalytically active, and the kinetic constants are nearly identical to the values obtained with the all-zinc-containing enzyme. The NMR spectrum of protein reconstituted with two 113Cd2+ ions per enzyme molecule exhibits cadmium resonances at 212 and 116 ppm downfield from Cd(ClO4)2. The two metal ions are, therefore, in significantly different chemical environments. These two binding sites have been designated the M alpha and M beta sites for the low- and high-field signals, respectively. Protein substituted with a single cadmium ion also shows two cadmium resonances, and thus one site is not completely filled prior to the binding of metal to the other site. The Cd/Zn hybrid protein shows a single cadmium resonance at 115 ppm, and thus the cadmium is occupying the M beta site while zinc is occupying the M alpha site. The positions of the observed chemical shifts for the two cadmium signals indicate that the ligands to both metals are composed of a mixture of oxygen and nitrogen atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the zinc binding site of bacterial phosphotriesterase.

The bacterial phosphotriesterase has been found to require a divalent cation for enzymatic activity. This enzyme catalyzes the detoxification of organophosphorus insecticides and nerve agents. In an Escherichia coli expression system significantly higher concentrations of active enzyme could be produced when 1.0 mM concentrations of Mn2+, Co2+, Ni2+, and Cd2+ were included in the growth medium. The isolated enzymes contained up to 2 equivalents of these metal ions as determined by atomic absorption spectroscopy. The catalytic activity of the various metal enzyme derivatives was lost upon incubation with EDTA, 1,10-phenanthroline, and 8-hydroxyquinoline-5-sulfonic acid. Protection against inactivation by metal chelation was afforded by the binding of competitive inhibitors, suggesting that at least one metal is at or near the active site. Apoenzyme was prepared by incubation of the phosphotriesterase with beta-mercaptoethanol and EDTA for 2 days. Full recovery of enzymatic activity could be obtained by incubation of the apoenzyme with 2 equivalents of Zn2+, Co2+, Ni2+, Cd2+, or Mn2+. The 113Cd NMR spectrum of enzyme containing 2 equivalents of 113Cd2+ showed two resonances at 120 and 215 ppm downfield from Cd(ClO4)2. The NMR data are consistent with nitrogen (histidine) and oxygen ligands to the metal centers.