Possible role of two phenylalanine residues in the active site of human cytidine deaminase

Site-directed mutagenesis on human cytidine deaminase (CDA)was employed to mutate specifically two highly conserved phenylalanineresidues, F36 and F137, to tryptophan; at the same time, theunique tryptophan residue present in the sequence at position113 was mutated to phenylalanine. These double mutations wereperformed in order to have for each protein a single tryptophansignal for fluorescence studies relative to position 36 or 137.The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F,showed by circular dicroism and thermal stability an overallstructure not greatly affected by the mutations. The titrationof Trp residues by N-bromosuccinimide (NBS) suggested that residueW113 of the wild-type CDA and W36 of mutant F36W/W113F are buriedin the tertiary structure of the enzyme, whereas the residueW137 of mutant F137W/W113F is located near the surface of themolecule. Kinetic experiments and equilibrium experiments withFZEB showed that the residue W113 seems not to be part of theactive site of the enzyme whereas the Phe/Trp substitution inF36W/W113F and F137W/W113F mutant enzymes had a negative effecton substrate binding and catalysis, suggesting that F137 andF36 of the wild-type CDA are involved in a stabilizing interactionbetween ligand and enzyme.