Identification of four 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 mutant proteins were obtained: C65A, C99A, C102A, E67D and E67Q. The three cysteine mutants were completely inactive, whereas E67D and E67Q showed a specific activity about 200- and 200000-fold lower, respectively, than the wild-type CDA. Zinc analysis revealed that only E67D, E67Q and C65A contained 1 mol Zn2+/mol subunit as in the wild-type CDA. Kinetic measurements with the specific carboxylic group reagent N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline performed on wild-type CDA suggest 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 to the sequence data. When p-hydroxymercuriphenyl sulfonate was used, nine 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 the maintenance of zinc in the active site is the formation of coordination between Cys99 and Cys102, whereas Cys65 could lead the zinc to the correct position and orientation within the active site.
Cambi
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Dipartimento di Biologia MCA, Università di Camerino, Italy.
Vincenzetti
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S
Neuhard
J
J
Costanzi
S
S
Natalini
P
P
Vita
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D019943:Amino Acid Substitution; D002384:Catalysis; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D016297:Mutagenesis, Site-Directed; D013438:Sulfhydryl Compounds / Q000737:chemistry; D015032:Zinc / Q000378:metabolism
D013438:Sulfhydryl Compounds; D003564:Cytidine Deaminase; D015032:Zinc