Purification of human cytidine deaminase: molecular and enzymatic characterization and inhibition by synthetic pyrimidine analogs.
Cytidine deaminase has been purified to homogeneity from human placenta by a rapid and efficient procedure consisting of affinity chromatography followed by hydrophobic interaction chromatography. The final enzyme preparation showed a specific activity of 64.1 units/mg, corresponding to about 46,000-fold purification with respect to the crude extract. The enzyme is a 52-kDa oligomeric protein composed of four apparently identical subunits. The acidic isoelectric point is 4.5. The enzyme's stability is strictly dependent on the presence of reducing agents. Amino acid analysis reveals the presence of five thiol groups per monomer which cannot be titrated by Ellman's reagent in the native enzyme. However, the presence of sulfhydryl groups involved in the catalytic activity was evidenced by the inhibition exerted by p-chloromercuribenzoate and heavy metal ions. In addition, the protection effected by the substrate against the p-chloromercuribenzoate inhibition and the competitive inhibition exerted by 5-(chloromercuri)cytidine suggest the presence of a thiol group(s) in the catalytic site of the enzyme. pH studies have shown that the rapid decline of activity occurring at pH 4.5 might result from the protonation of the pyrimidine ring at the N-3 position. The enzyme catalyzes the deamination of cytidine, deoxycytidine, and several analogs, including antineoplastic agents, thus abolishing their pharmacological activity. Therefore, several pyrimidine nucleoside analogs have been tested as potential inhibitors of the enzyme. The competitive inhibition exerted by cytidine analogs having the ribose moiety replaced by aliphatic chains is interesting.
Dipartimento di Biologia M.C.A., Università di Camerino, Italy.
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D000596:Amino Acids / Q000032:analysis; D003564:Cytidine Deaminase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010920:Placenta / Q000201:enzymology; D011743:Pyrimidines / Q000138:chemical synthesis / Q000494:pharmacology*; D013439:Sulfhydryl Reagents; D013696:Temperature
D000596:Amino Acids; D008670:Metals; D011743:Pyrimidines; D013439:Sulfhydryl Reagents; D003564:Cytidine Deaminase