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 phospho diesterase (cGI-PDE) is the predominant platelet enzyme hydrolysing cA MP. The pH-rate profile plot for this enzyme yields pK(a) values of 6. 5 and 9.0, consistent with histidine and cysteine residues respectivel y. Diethyl pyrocarbonate (DEP) inactivates cGI-PDE in a time- and conc entration-dependent manner, and this effect was rapidly reversed by hy droxylamine. It was estimated that 2 mol of histidine residues per mol of enzyme were responsible for the loss of catalytic activity, as ded uced from the correlation of the difference spectrum at 240 nm of the DEP-modified cGI-PDE with the enzymic 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 DE P, NEM and DTNB, suggesting the presence of histidine and cysteine res idues at the active site of cGI-PDE. [C-14]DEP incorporation in the pr esence of AMP or cGMP indicates the protection of two histidine residu es by each nucleotide. These residues are different for each agent, si nce the combination of AMP and cGMP protects four histidine residues. [H-3]NEM incorporation showed that 1 mol of cysteine per mol of cGI-PD E was protected by AMP, but not by cGMP. We conclude that cGI-PDE poss esses two essential histidine residues for activity, two additional hi stidines for cGMP inhibition, and one cysteine residue at the active s ite.