IDENTIFICATION OF 4 AMINO-ACID-RESIDUES ESSENTIAL FOR CATALYSIS IN HUMAN CYTIDINE DEAMINASE BY SITE-DIRECTED MUTAGENESIS AND CHEMICAL MODIFICATIONS

By site-directed mutagenesis on human cytidine deaminase (CDA), five m utant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The th ree cysteine mutants were completely inactive, whereas E67D and E67Q s howed a specific activity about 200- and 200 000-fold lower, respectiv ely, than the wild-type CDA. Zinc analysis revealed that only E67D, E6 7Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA. Kinetic measurements with the specific carboxylic group reagent N-etho xycarbonyl-3-ethoxy-1,2-dihydroquinoline performed on wild-type CDA su ggest that Glu67 is essential for the catalytic process. Furthermore, when both native and denatured CDA was titrated with 5,5'-dithiobis(2- nitrobenzoic acid) six sulfhydryl groups were detected, whereas in the denatured and reduced enzyme nine such groups were found, according t o the sequence data. When p-hydroxymercuriphenyl sulfonate was used, n ine sulfhydryl groups were detectable and the release of 1 mol of zinc per mole of CDA subunit was revealed by the metal indicator dye 4-(2- pyridylazo)resorcinol. It seems plausible that the limiting step for t he maintenance of zinc in the active site is the formation of coordina tion between Cys99 and Cys102, whereas Cys65 could lead the zinc to th e correct position and orientation within the active site.