Cytidine deaminases from B-subtilis and E-coli: Compensating effects of changing zinc coordination and quaternary structure

Cytidine deaminase from E. coli is a dimer of identical subunits (M-r = 31 540), each containing a single zinc atom. Cytidine deaminase from B. subtil is is a tetramer of identical subunits (M-r = 14 800). After purification f rom an overexpressing strain, the enzyme from B, subtilis is found to conta in a single atom of zinc per enzyme subunit by flame atomic absorption spec troscopy. Fluorescence titration indicates that each of the four subunits c ontains a binding site for the transition state analogue inhibitor 5-fluoro -3,4-dihydrouridine. A region of amino acid sequence homology, containing r esidues that are involved in zinc coordination in the enzyme from E, coli, strongly suggests that in the enzyme from B. subtilis, zinc is coordinated by the thiolate side chains of three cysteine residues (Cys-53, Cys-86, and Cys-89) [Song, B, H., and Neuhard, J. (1989) Mol. Gen. Genet. 216, 462-468 ]. This pattern of zinc coordination appears to be novel for a hydrolytic e nzyme, and might be expected to reduce the reactivity of the active site su bstantially compared with that of the enzyme from E. coli (His-102, Cys-129 , and Cys-132). Instead, the B, subtilis and E. coli enzymes are found to b e similar in their activities, and also in their relative binding affinitie s for a series of structurally related inhibitors with binding affinities t hat span a range of 6 orders of magnitude. In addition, the apparent pK(a) value of the active site is shifted upward by less than 1 unit. Sequence al ignments, together with model building, suggest one possible mechanism of c ompensation.