UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl 2 or MgCl 2 , the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (K m = 0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S 0.5 = 9.4 + − 0.7 mM and n = 1.9 + − 0.1 in the presence of MgCl 2 ). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient.The k cat (175 + − 13 s −1 in the presence of MgCl 2 ) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP.

Interleukin-1β-converting enzyme is a member of a family of human cysteine proteases with specificity for aspartic acid, which have been named caspases. Within this family of enzymes, transcript X (TX) and transcript Y (TY) (caspases 4 and 5, respectively) are very similar to ICE (caspase 1) and form the ICE subfamily. Given the high degree of conservation in the sequences of these proteases (more than 50% amino acid identity in the mature enzymes), it was of interest to examine whether they shared similar substrate specificities. The three enzymes, ICE, TX and TY, were therefore expressed in baculovirus-infected insect cells, as 30-kDa proteins lacking the propeptide. Automaturation into p20 and p10 subunits occured within the cells. Active ICE, TX and TY were collected in the cell culture supernatants. In addition, their production induced the activation of an endogenous 32-kDa putative cysteine protease (CPP32) like caspase. T7-tagged ICE, TX and TY were purified by immunoaffinity and tested for their catalytic efficiency on YVAD-containing synthetic substrates and on the ICE natural substrate, pro-interleukin-1β. TX cleaved the same synthetic substrates as ICE (K m of 90 µM and k cat of 0.4 s Ϫ1 for Suc-YVAD-NH-Mec, where Suc represents succinyl and NH-Mec represents amino-4-methylcoumarin) and could cleave pro-interleukin-1β into the same peptides as ICE but less efficiently. On the other hand, TY showed very little efficacy on the different ICE substrates (K m of 860 µM for Suc-YVAD-NH-Mec). These results show that the ICE/TX/TY subfamily has functional heterogeneity and that ICE remains the preferred enzyme for pro-interleukin-1β cleavage.Keywords : caspase ; cysteine protease ; interleukin-1β-converting enzyme homolog; interleukin-1β.Interleukin-1β (IL-1β)-converting enzyme (ICE) is a cyste-tein with death-domain-like ICE2) [3, 4]. A second subfamily is ine protease responsible for the cleavage of the inflammatory composed of caspase 1 (ICE), caspase 4 [transcript X (TX)/ICE cytokine proIL-1β to its biologically active form [ [7,8]. The protein implicated in apoptosis during the development of third group has only one member, caspase 2 (ICH1) [3]. All the Caenorhabditis elegans. Since then, numerous homologs of ICE proteins of the family share characteristic properties based on and Ced3 have been identified. They are designated the caspase sequence comparisons: conserved QACXG motif around the family to indicate that they are all cysteine proteases and cleave active-site cysteine ; specificity for aspartic acid at P1; and a at the C-terminus of an aspartic acid residue [3]. The human heterodimeric structure after activation by cleavage of a precurenzymes are classified into three groups according to similarities sor at aspartic acid residues. Automaturation has been demonin amino acid sequences. One group, related to Ced3, is called strated in vitro for ICE p45 zymogen [9, 10] but its physiological the 32-kDa putative cysteine protease (CCP32) subfamily and relevance remains to be established [11]. Sequ...