Escherichia coli cytosine deaminase: the kinetics and thermodynamics for binding of cytosine to the apoenzyme and the Zn2+ holoenzyme are similar

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Abstract

Recombinant Escherichia coli cytosine deaminase is purified as a mixture of Zn2+ and Fe2+ forms of the enzyme. Fe2+ is removed readily by o-phenanthroline to yield apoenzyme (apoCDase) that contains <0.2 mol of Zn2+per mol of subunit. ApoCDase was efficiently reconstituted to Zn2+CDase by treatment with ZnCl2. The interaction of cytosine with apoCDase and Zn2+CDase was investigated at pH 7.5 and 25°C by monitoring changes in intrinsic protein fluorescence. The values for the kinetic data K1, k2, and k3 for Zn2+CDase were 0.25 mM, 80 s−1, and 38 s−1, respectively. The value for k−2 was statistically indistinguishable from zero. The analogous values for K1, k2, and k−2, (k3=0) for apoCDase were 0.157 mM, 186 s−1 and ∼0.8 s−1, respectively. The overall dissociation constant of apoCDase for cytosine was 0.00069 mM, whereas the Km of Zn2+CDase for cytosine was 0.20 mM. The pre-steady state phase of the reaction was associated with an absorbance increase at 280 nm that was attributed to solvent perturbation of the spectrum of cytosine or enzyme. Formation of the Fe2+CDase–cytosine complex was too rapid to monitor by these techniques.

Introduction

Cytosine deaminase (CDase, EC 3.5.4.1) catalyzes the deamination of cytosine and 5-fluorocytosine to uracil and 5-fluorouracil, respectively [1], [2]. Because mammalian cells do not have detectable CDase activity, selective delivery of CDase to malignant cells has been suggested as a novel means for in situ generation of 5-fluorouracil from orally dosed 5-fluorocytosine to treat cancer [3], [4], [5], [6], [7], [8]. CDase has been purified from a genetically engineered strain of Escherichia coli that over expressed the codBA operon on a multi-copy plasmid [9]. The isolated CDase is a mixture of the Zn2+-form (Zn2+CDase) and the Fe2+-form (Fe2+CDase) of the enzyme [9]. Fe2+ is reversibly removed from the enzyme by o-phenanthroline. Cytosine deaminating activity is completely restored to apoenzyme by adding Fe2+ [9]. A similar mixture of metal cofactors has been reported for creatinine iminohydrolyase (EC 3.5.4.21) from Flavobacterium filamentosum [10]1.

The affinity of deaminases for metal cofactors varies greatly. For example, calf intestinal and human adenosine deaminases bind Zn2+ sufficiently tightly that supplemental Zn2+ is not required in the purification buffer for maximal activity, whereas the recombinant mouse enzyme requires supplemental Zn2+ in the purification buffer for recovery of maximal adenosine deaminase activity [11], [12]. Reversible formation of the apoenzyme of many deaminases is hampered by the harsh conditions required for resolution. In particular, E. coli cytidine deaminase binds the Zn2+ cofactor very tightly [13]). However, mutation of His102 to an Asp (H102N) results in a mutant enzyme, which retained catalytic activity, that could be reversibly resolved into apoenzyme by dialysis against EDTA [13]. The product uridine bound the mutant apoenzyme with approximately 10-fold greater affinity than the holoenzyme. Thus, substrate affinity might actually be enhanced in the absence of the metal cofactor. The availability of apoCDase and the opportunity to reconstitute quantitatively the apoenzyme with Zn2+ presented the reagents for a detailed comparison of the thermodynamic and kinetic parameters for cytosine binding to the apoenzyme and Zn2+-holoenzyme. Steady-state and pre-steady-state techniques were used herein to investigate the interaction of cytosine with apoCDase and Zn2+CDase. In summary, Zn2+CDase formed a transient complex with cytosine that had reduced intrinsic protein fluorescence. Pre-steady-state and steady-state kinetic results were consistent with the participation of this intermediate in catalysis. The value of Km of Zn2+CDase for cytosine was over 200-fold greater than the estimated value of the Kd of Zn2+CDase and apoCDase for cytosine.

Section snippets

Materials

Tris, o-phenanthroline, cytosine, 5-fluorocytosine, 5-azacytosine, 6-azacytosine, 2-thiocytosine, creatinine, 1-methylcytosine, 3-methylcytosine, 5-methylcytosine 4-aminopyrimidine, 2-hydroxypyrimidine, and uracil were from Sigma Chemical Co. (St. Louis, MO). 4-Thiouracil was from Aldrich Chemical Co. Whatman DE-52 anion-exchange resin was purchased from Whatman. Bio-Gel P-6 was from Bio-Rad. Reagent grade FeSO4 and ZnCl2 were from Mallinckrodt. [6-3H]cytosine (20 Ci/mmol) was from Moravek

Reconstitution of apoCDase with Zn2+

Freshly isolated CDase contains tightly bound Fe2+ and Zn2+ in the ratio of approximately 4:1. Fe2+ can be removed from this enzyme by treatment with o-phenanthroline with concomitant loss of catalytic activity [9]. Zn2+ was not removed significantly by o-phenanthroline treatment [9]. Most importantly, the catalytic activity lost upon removal of Fe2+ was quantitatively recovered by addition of Fe2+ to the apoenzyme. Titration of the apoenzyme by Fe2+ yielded a stoichiometry of 0.8 Fe2+ sites

Discussion

Zielke and Suelter demonstrated in 1971 that AMP deaminase was a zinc metalloenzyme [16]. An apoenzyme preparation of AMP deaminase, which contained less that 10% of the catalytic activity of the holoenzyme, was made by treatment of the holoenzyme with 8-hydroxyquinoline-5-sulfonate. The apoenzyme could be reconstituted with Zn2+, Co2+, Mn2+ or Fe2+ to yield a holoenzyme with similar catalytic efficiency [16]. More recently, Wilson et al. [17] have demonstrated that adenosine deaminase contains

Acknowledgements

The author gratefully acknowledges Dr. E. Austin for making the cytosine deaminase overproducing strain of E. coli available and for her help in growing the bacteria. Informative discussions with Dr. S. Short concerning biochemistry of deaminases were also greatly appreciated.

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