Functional properties of subunit interactions in human cytidine deaminase.
An intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In the presence of SDS, wild-type human CDA dissociates into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. Circular dichroism measurements show that the secondary/tertiary structure organization of each subunit is unaffected by the SDS concentration, while the mutation Phe/Trp causes weakening in quaternary structure. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavours dissociation of the tetramer into subunits in the wild-type CDA, but not in mutant enzyme F137W/W113F. The absence of tyrosine fluorescence and the much higher quantum yield of the double mutant protein spectrum suggest the occurrence of an energy transfer effect between the protein subunits. This assumption is confirmed by the crystallographic studies on B.subtilis in which it is shown that three different subunits concur with the formation of each of the four active sites and that F125, homologous to the human CDA F137, is located at the interface between two different subunits contributing to the formation of active site.
Dipartimento di Scienze Veterinarie, Università di Camerino, Italy. silvia.vincenzetti@unicam.it
Silvia
S
Vincenzetti
Giampiero
G
De Sanctis
Stefano
S
Costanzi
Gloria
G
Cristalli
Pierluigi
P
Mariani
Giampiero
G
Mei
Jan
J
Neuhard
Paolo
P
Natalini
Valeria
V
Polzonetti
Alberto
A
Vita
D002942:Circular Dichroism; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D009154:Mutation; D010649:Phenylalanine / Q000235:genetics / Q000502:physiology; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D012967:Sodium Dodecyl Sulfate; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D014364:Tryptophan / Q000235:genetics / Q000502:physiology
D012967:Sodium Dodecyl Sulfate; D010649:Phenylalanine; D014364:Tryptophan; D003564:Cytidine Deaminase