Deoxycytidylate deaminase. Purification and some properties of the enzyme isolated from human spleen.

Deoxycytidylate deaminase (dCMP aminohydrolase, EC 3.5.4.12) from human spleen has been purified 2,515fold to apparent homogeneity. The native M, of the enzyme is 109,500 and the Stokes radius is 43 A as determined by gel filtration. The subunit M, = 53,000 as determined by sodium dodecyl sulfate-gel electrophoresis, suggesting that the enzyme is a dimer in its native state. Human spleen deoxycytidylate deaminase has a broad pH optimum between 6.5 and 7.3. With isoelectric focusing, the enzyme demonstrated a single form with an isoelectric point of 5.10. Studies of the kinetic behavior of the purified enzyme showed a sigmoidal initial velocity versus substrate concentration curve and highly specific regulation by allosteric effectors dCTP and ‘ITP. The allosteric activator dCTP changed the sigmoidal initial velocity versus substrate concentration curve to a hyperbolic. one. The apparent activation K, for dCTP was 0.25 PM. 1-fi-o-Arabinohranosylcytosine S’-triphosphate, CTP, dCDP, and CDP also stimulated enzyme activity but the enzyme had a lower affinity for these nucleotides, and the respective apparent activation K, values ranged from 40 to 70 PM. TTP was found to have a dual effect on human spleen deoxycytidylate deaminase, stimulating the enzyme by up to 1.7-fold at a concentration of 0.25 p~ but inhibiting the enzyme at higher concentrations, with 85% of enzyme activity inhibited at 15 PM ITP. Human spleen deoxycytidylate deaminase exhibited a high degree of specificity of dCMP. CMP was a poor substrate for the enzyme, while the I-fi-D-arabinose analogue ara-CMP was an effective substrate in the presence of dCTP concentrations exceeding 50 PM. A number of purine and pyrimidine nucleotides were competitive inibitors of the enzyme, of which dGMP (Ki, 175 PM) was the most effective while the reaction product dUMP (Ki, 1,150 p ~ ) and ‘I”P (Ki, 1,400 PM) were the least inhibitory.