Protein Engineering vol. 16 no. 12 pp. 1055-1061, 2003
© 2003 Oxford University Press
Functional properties of subunit interactions in human cytidine deaminase
1Dipartimento di Scienze Veterinarie, 3Dipartimento di Biologia MCA, 4Dipartimento di Scienze Chimiche, 7Dipartimento di Scienze Morfologiche e Biochimiche Comparate, Università di Camerino, 5Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata and INFM, Italy and 6Institute of Molecular Biology, University of Copenhagen, Denmark
2 To whom correspondence should be addressed. e-mail: silvia.vincenzetti{at}unicam.it
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.
Received February 10, 2003; revised October 14, 2003;; accepted October 21, 2003
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