Structural and Catalytic Properties of CMP Kinase fromBacillus subtilis:A Comparative Analysis with the Homologous Enzyme fromEscherichia coli

Abstract

CMP kinases fromBacillus subtilisand fromEscherichia coliare encoded by thecmkgene (formerly known asjofCinB. subtilisand asmssAinE. coli). Similar in their primary structure (43% identity and 67% similarity in aminoacid sequence), the two proteins exhibit significant differences in nucleotide binding and catalysis. ATP, dATP, and GTP are equally effective as phosphate donors withE. coliCMP kinase whereas GTP is a poor substrate withB. subtilisCMP kinase. While CMP and dCMP are the best phosphate acceptors of both CMP kinases, the specific activity with these substrates and ATP as donor are 7- to 10-fold higher in theE. colienzyme; the relativeVmvalues with UMP and CMP are 0.1 for theB. subtilisCMP kinase and 0.01 for theE. colienzyme. CMP increased the affinity ofE. coliCMP kinase for ATP or for the fluorescent analog 3′-anthraniloyl dATP by one order of magnitude but had no effect on theB. subtilisenzyme.The differences in the catalytic properties ofB. subtilisandE. coliCMP kinases might be reflected in the structure of the two proteins as inferred from infrared spectroscopy. Whereas the spectrum ofB. subtilisCMP kinase is dominated by a band at 1633 cm−1(representing β type structures), the spectrum of theE. colienzyme is dominated by two bands at 1653 and 1642 cm−1associated with α-helical and unordered structures, respectively. CMP induced similar spectral changes in both proteins with a rearrangement of some of the β-structures. ATP increases the denaturation temperature ofB. subtilisCMP kinase by 9.3°C, whereas in the case of theE. colienzyme, binding of ATP has only a minor effect.