Abstract
We have used an incomplete factorial design (Carter, C. W., and Carter, C. W., Jr. (1979) J. Biol. Chem. 254, 12219-12223) to find conditions for growing high quality crystals of Escherichia coli cytidine deaminase (EC 3.5.4.5). Crystals grow at pH 6.0 in hanging or sitting drops with either 1.6 M ammonium sulfate or 2.4-2.5 M sodium phosphate as precipitant. Both conditions produce crystals with identical morphologies and unit cell constants. The space group is P3(1)21 (or its enantiomorph P3(2)21), and the unit cell constants are a = b = 120.3 A, c = 78.4 A. The asymmetric unit is most reasonably one dimer of 66,000 Mr. The crystal size is very dependent on the supersaturation ratio, S = [initial protein concentration]/[equilibrium protein concentration], exhibiting a maximum at S = 7.7. The largest crystals diffract to at least 2.5 A and have a lifetime of 4 to 5 days in the x-ray beam at room temperature. The enzyme in these crystals is complexed with the transition state analog inhibitor 1-(beta-D-ribofuranosyl)-5-fluoropyrimidin-2-one (5-fluoropyrimidin-2-one riboside). We have collected data from parent crystals and from a heavy atom derivative in which the transition state analog is replaced by the active site directed inhibitor 5-(chloromercuri)cytidine.
Highlights
Incomplete Factorial Search for Conditions Leading to High Quality Crystals of Escherichia coliCytidine Deaminase Complexed toa Transition State Analog Inhibitor*
High affinity transition state analog inhibitors have been discovered for both cytidine deaminase (Cohen and Wolfenden, 1971; Marquez et al, 1980; Liu et al, 1984; Ashley and Bartlett, 1984a,1984b;Kim et al, 1986) and adenosine deaminase (Evans andWolfenden, 1970;Ohno etal., 1974; Wood et al, 1974; Wentworth and Wolfenden, 1977)
Powerful inhibition by these compounds is believed to be due to a resemblance between bound inhibitors and the transition state for enzyme-assisted displacement of ammonia,in which either water or a nucleophilic group on the Cytidine deaminase (EC 3.5.4.5) catalyzes the hydrolytic enzyme could serve as the attacking nucleophile
Summary
Incomplete Factorial Search for Conditions Leading to High Quality Crystals of Escherichia coliCytidine Deaminase Complexed toa Transition State Analog Inhibitor*. It is important to know the chemical and structural basis for inhibition of this enzyme.We describe here astatistically designed search which has identified conditions for growing high quality crystals of E. coli cytidine deaminase complexed to the transition state analog inhibitor 5-fluoropyrimidin-2one riboside.
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