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.
Ghazaleh
F A
FA
Departmento de Bioquimica Medica, Universidade Federal do Rio de Janeiro, Brazil.
Omburo
G A
GA
Colman
R W
RW
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000201:enzymology*; D002384:Catalysis; D000242:Cyclic AMP / Q000378:metabolism*; D006152:Cyclic GMP / Q000494:pharmacology*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D003545:Cysteine / Q000737:chemistry; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*
D010726:Phosphodiesterase Inhibitors; D000249:Adenosine Monophosphate; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; D006152:Cyclic GMP; D003545:Cysteine; D004047:Diethyl Pyrocarbonate; D005033:Ethylmaleimide