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 phenylalanine residues, F36 and F137, to tryptophan; at the same time, the unique tryptophan residue present in the sequence at position 113 was mutated to phenylalanine. These double mutations were performed in order to have for each protein a single tryptophan signal 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 overall structure not greatly affected by the mutations. The titration of Trp residues by N-bromosuccinimide (NBS) suggested that residue W113 of the wild-type CDA and W36 of mutant F36W/W113F are buried in the tertiary structure of the enzyme, whereas the residue W137 of mutant F137W/W113F is located near the surface of the molecule. Kinetic experiments and equilibrium experiments with FZEB showed that the residue W113 seems not to be part of the active site of the enzyme whereas the Phe/Trp substitution in F36W/W113F and F137W/W113F mutant enzymes had a negative effect on substrate binding and catalysis, suggesting that F137 and F36 of the wild-type CDA are involved in a stabilizing interaction between ligand and enzyme.
Vincenzetti
S
S
Dipartimento di Scienze Veterinarie, Università di Camerino, via Circonvallazione 93-95, 62024 Matelica, Italy.
Cambi
A
A
Maury
G
G
Bertorelle
F
F
Gaubert
G
G
Neuhard
J
J
Natalini
P
P
Salvatori
D
D
De Sanctis
G
G
Vita
A
A
D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli; D005453:Fluorescence; D006801:Humans; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000378:metabolism*; D011741:Pyrimidine Nucleosides / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism
D011741:Pyrimidine Nucleosides; D010649:Phenylalanine; C059356:5-fluoropyrimidin-2-one beta-ribofuranoside; D014364:Tryptophan; D003564:Cytidine Deaminase