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• W2081482806 abstract "The effect of spermine on the inhibition of peptide-bond formation by clindamycin, an antibiotic of the Macrolide-Lincosamide-StreptograminsB family, was investigated in a cell-free system derived from Escherichia coli. In this system peptide bond is formed between puromycin, a pseudo-substrate of the A-site, and acetylphenylalanyl-tRNA, bound at the P-site of poly(U)-programmed 70 S ribosomes. Biphasic kinetics revealed that one molecule of clindamycin, after a transient interference with the A-site of ribosomes, is slowly accommodated near the P-site and perturbs the 70 S/acetylphenylalanyl-tRNA complex so that a peptide bond is still formed but with a lower velocity compared with that observed in the absence of the drug. The above mechanism requires a high temperature (25 °C as opposed to 5 °C). If this is not met, the inhibition is simple competitive. It was found that at 25 °C spermine favors the clindamycin binding to ribosomes; the affinity of clindamycin for the A-site becomes 5 times higher, whereas the overall inhibition constant undergoes a 3-fold decrease. Similar results were obtained when ribosomes labeled with N1-azidobenzamidinospermine, a photo-reactive analogue of spermine, were used or when a mixture of spermine and spermidine was added in the reaction mixture instead of spermine alone. Polyamines cannot compensate for the loss of biphasic kinetics at 5 °C nor can they stimulate the clindamycin binding to ribosomes. Our kinetic results correlate well with photoaffinity labeling data, suggesting that at 25 °C polyamines bound at the vicinity of the drug binding pocket affect the tertiary structure of ribosomes and influence their interaction with clindamycin. The effect of spermine on the inhibition of peptide-bond formation by clindamycin, an antibiotic of the Macrolide-Lincosamide-StreptograminsB family, was investigated in a cell-free system derived from Escherichia coli. In this system peptide bond is formed between puromycin, a pseudo-substrate of the A-site, and acetylphenylalanyl-tRNA, bound at the P-site of poly(U)-programmed 70 S ribosomes. Biphasic kinetics revealed that one molecule of clindamycin, after a transient interference with the A-site of ribosomes, is slowly accommodated near the P-site and perturbs the 70 S/acetylphenylalanyl-tRNA complex so that a peptide bond is still formed but with a lower velocity compared with that observed in the absence of the drug. The above mechanism requires a high temperature (25 °C as opposed to 5 °C). If this is not met, the inhibition is simple competitive. It was found that at 25 °C spermine favors the clindamycin binding to ribosomes; the affinity of clindamycin for the A-site becomes 5 times higher, whereas the overall inhibition constant undergoes a 3-fold decrease. Similar results were obtained when ribosomes labeled with N1-azidobenzamidinospermine, a photo-reactive analogue of spermine, were used or when a mixture of spermine and spermidine was added in the reaction mixture instead of spermine alone. Polyamines cannot compensate for the loss of biphasic kinetics at 5 °C nor can they stimulate the clindamycin binding to ribosomes. Our kinetic results correlate well with photoaffinity labeling data, suggesting that at 25 °C polyamines bound at the vicinity of the drug binding pocket affect the tertiary structure of ribosomes and influence their interaction with clindamycin. Lincomycin and clindamycin (Fig. 1) are lincosamides, still used as therapeutic agents in human diseases and some animal infections. Clindamycin, a semisynthetic derivative of lincomycin (7(S)-chloro-7-deoxylincomycin), is usually more active than the parent compound in the treatment of bacterial infections, in particular those caused by anaerobic species (1Spižek J. Řezanka T. Appl. Microbiol. Biotechnol. 2004; 64: 455-464Crossref PubMed Scopus (142) Google Scholar). Lincosamides act on the large ribosomal subunit and share an overlapping binding site with macrolides, streptogramins B, chloramphenicol, and other antibiotics affecting the PTase 2The abbreviations used are: PTase, peptidyltransferase; ABA, N1-azidobenzamidino; AcPhe, acetylphenylalanyl; DR, double reciprocal. center, as deduced from competition experiments and cross-resistance data (for review, see Ref. 2Roberts M.C. Mol. Biotechnol. 2004; 28: 47-62Crossref PubMed Scopus (93) Google Scholar) as well as from two-dimensional transferred nuclear Overhauser effect spectroscopy analysis (3Verdier L. Bertho G. Gharbi-Benarous J. Girault J.-P. Bioorg. Med. Chem. 2000; 8: 1225-1243Crossref PubMed Scopus (26) Google Scholar). In agreement with most of these observations, chemical footprinting analysis (4Douthwaite S. Nucleic Acids Res. 1992; 20: 4717-4720Crossref PubMed Scopus (68) Google Scholar, 5Kehrenberg C. Schwarz S. Jacobsen L. Hansen L.H. Vester B. Mol. Microbiol. 2005; 57: 1064-1073Crossref PubMed Scopus (250) Google Scholar) and crystallographic studies (6Schlünzen F. Zarivach R. Harms J. Bashan A. Tocilj A. Albrecht R. Yonath A. Franceschi F. Nature. 2001; 413: 814-821Crossref PubMed Scopus (890) Google Scholar, 7Tu D. Blaha G. Moore P.B. Steitz T.A. Cell. 2005; 121: 257-270Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) have revealed that lincosamides interact with both the A-site and the P-site on the 50 S ribosomal subunit and would be expected to hamper the positioning of aminoacyl moieties of tRNAs at both sites. This last view is consistent with earlier binding studies investigating the competition for binding to ribosomes between lincomycin and 3′-terminus pentanucleotide fragments from N-AcLeu-tRNALeu, Leu-tRNALeu, and Phe-tRNAPhe (8Celma M.L. Monro R.E. Vázquez D. FEBS Lett. 1971; 13: 247-251Crossref PubMed Scopus (69) Google Scholar, 9Cěrná J. Rychlík I. Biochim. Biophys. Acta. 1972; 287: 292-300Crossref PubMed Scopus (13) Google Scholar) as well as with the finding that lincosamides stimulate oligopeptidyl-tRNA dissociation from ribosomes (10Menninger J.R. Coleman R.A. Antimicrob. Agents Chemother. 1993; 37: 2027-2029Crossref PubMed Scopus (24) Google Scholar). Tenson et al. (11Tenson T. Lovmar M. Ehrenberg M. J. Mol. Biol. 2003; 330: 1005-1014Crossref PubMed Scopus (324) Google Scholar) suggested that lincosamides bind to ribosomes allowing a space of ∼4.6 Å for nascent peptide progression toward the exit tunnel. Further peptidyl-transfer is inhibited by steric hindrance, and this inhibition eventually leads to peptidyl-tRNA dissociation. Therefore, an increased probability of ribosome drop-off can be caused by enhanced peptidyl-tRNA dissociation, decreased PTase activity, or a combination of both. In consequence, termination is also affected by both lincomycin and clindamycin (12Polacek N. Gomez M.J. Ito K. Xiong L. Nakamura Y. Mankin A. Mol. Cell. 2003; 11: 103-112Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In addition to the inhibitory effect on protein synthesis, each lincosamide exhibits a specific inhibitory effect on the 50 S-subunit formation, an activity shared with 16-membered macrolide and streptogramin B antibiotics (13Champney W.S. Tober C.L. Curr. Microbiol. 2000; 41: 126-135Crossref PubMed Scopus (52) Google Scholar). Molecular modeling approaches have been used in at least three previous attempts to establish structural relationships between lincosamides and residues in the 3′-terminus of aminoacyl-tRNAs or hypothetic transition states and intermediates in protein biosynthesis (14Harris R.J. Symons R.H. Bioorg. Chem. 1973; 2: 266-285Crossref Scopus (29) Google Scholar, 15Cheney B.V. J. Med. Chem. 1974; 17: 590-599Crossref PubMed Scopus (19) Google Scholar, 16Fitzhugh A.L. Bioorg. Med. Chem. Lett. 1998; 8: 87-92Crossref PubMed Scopus (15) Google Scholar). These hypotheses were later either contradicted by crystallographic evidence or never verified. Despite considerable interest for elucidation of the lincosamide binding site in ribosomes, kinetic studies concerning the inhibitory effect of these drugs on PTase activity are scant and exclusively focused on lincomycin. This drug cannot affect peptidyl-puromycin synthesis on native polyribosomes but inhibits transpeptidation with washed ribosomes and diverse synthetic peptidyl donors (17Pestka S. J. Biol. Chem. 1972; 247: 4669-4678Abstract Full Text PDF PubMed Google Scholar). Besides competitive kinetics (18Hausner T.-P. Geigenmüller U. Nierhaus K.H. J. Biol. Chem. 1988; 263: 13103-13111Abstract Full Text PDF PubMed Google Scholar, 19Kallia-Raftopoulos S. Kalpaxis D.L. Coutsogeorgopoulos C. Arch. Biochem. Biophys. 1992; 298: 332-339Crossref PubMed Scopus (11) Google Scholar), there is also evidence for a mixed-noncompetitive mode of action (20Kallia-Raftopoulos S. Kalpaxis D.L. Coutsogeorgopoulos C. Mol. Pharmacol. 1994; 46: 1009-1014PubMed Google Scholar) depending on the inhibitor concentration and the buffer system used. From kinetic and binding studies, it has been demonstrated that both monovalent and Mg2+ ions are essential components for the PTase activity and the binding of lincomycin to ribosomes (20Kallia-Raftopoulos S. Kalpaxis D.L. Coutsogeorgopoulos C. Mol. Pharmacol. 1994; 46: 1009-1014PubMed Google Scholar, 21Chang F.N. Weisblum B. Biochemistry. 1967; 6: 836-843Crossref PubMed Scopus (34) Google Scholar, 22Vogel Z. Vogel T. Zamir A. Elson D. J. Mol. Biol. 1971; 60: 339-346Crossref PubMed Scopus (39) Google Scholar). This is in agreement with the finding that lincomycin forms metal complexes in solution (23Gaggelli E. Gaggelli N. Valensin D. Valensin G. Jezowska-Bojczuk M. Kozlowski H. Inorg. Chem. 2002; 41: 1518-1522Crossref PubMed Scopus (13) Google Scholar). In fact, recent crystallographic studies (6Schlünzen F. Zarivach R. Harms J. Bashan A. Tocilj A. Albrecht R. Yonath A. Franceschi F. Nature. 2001; 413: 814-821Crossref PubMed Scopus (890) Google Scholar, 7Tu D. Blaha G. Moore P.B. Steitz T.A. Cell. 2005; 121: 257-270Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar) do not detect any metal ion involved in the binding of clindamycin; however, one Mg2+ ion is displaced upon clindamycin binding. In addition to monovalent and divalent ions, polyamines are also essential for establishing an optimum ionic environment for ribosomal activity (24Bartetzko A. Nierhaus K.H. Methods Enzymol. 1988; 164: 650-658Crossref PubMed Scopus (67) Google Scholar). Interestingly, polycations sensitize enteric bacteria to clindamycin (25Vaara M. Vaara T. Antimicrob. Agents Chemother. 1983; 24: 107-113Crossref PubMed Scopus (161) Google Scholar). Consequently, a prerequisite when performing in vitro experiments is to take into account the physiological conditions and to try as closely as possible to imitate them. Unfortunately, most of the kinetic studies concerning the binding of lincosamides to ribosomes and the inhibition of PTase by these drugs have been performed exclusively in the presence of conventional buffers. Moreover, they have been analyzed on the assumption that the equilibria involved in the interaction of the drug with the ribosome are attained instantaneously. In fact, there is experimental evidence that the lincosamide-ribosome interactions display biphasic kinetics (3Verdier L. Bertho G. Gharbi-Benarous J. Girault J.-P. Bioorg. Med. Chem. 2000; 8: 1225-1243Crossref PubMed Scopus (26) Google Scholar, 19Kallia-Raftopoulos S. Kalpaxis D.L. Coutsogeorgopoulos C. Arch. Biochem. Biophys. 1992; 298: 332-339Crossref PubMed Scopus (11) Google Scholar). Contradicting observations have been published concerning the inhibitory power of clindamycin. NMR studies revealed that this antibiotic compared with lincomycin exhibits higher affinity for the ribosome (3Verdier L. Bertho G. Gharbi-Benarous J. Girault J.-P. Bioorg. Med. Chem. 2000; 8: 1225-1243Crossref PubMed Scopus (26) Google Scholar), in agreement with in vivo studies carried out in common pathogenic bacteria including Escherichia coli (1Spižek J. Řezanka T. Appl. Microbiol. Biotechnol. 2004; 64: 455-464Crossref PubMed Scopus (142) Google Scholar). In another study, however, the in vitro affinity of lincomycin to E. coli ribosomes was reported to be higher than that of clindamycin (4Douthwaite S. Nucleic Acids Res. 1992; 20: 4717-4720Crossref PubMed Scopus (68) Google Scholar). Kinetic studies of clindamycin interaction with functional ribosomal complexes have never been performed in the past nor has the effect of polyamines on this interaction been investigated. In the present study we examine the interaction of clindamycin with a post-translocation complex of poly(U)-programmed ribosomes isolated from E. coli cells. We employ an experimental approach by utilizing a series of polyamine buffers and by analyzing the peptide bond formation in the presence of clindamycin as a pseudo-first-order reaction. This approach allows us to have a picture of the entire course of the reaction. The use of polyamine buffers not only better resembles the physiological ionic environment but also gives us the opportunity to kinetically reveal the contribution of polyamines in the interaction of clindamycin with ribosomes. Materials—Puromycin dihydrochloride (disodium salt), clindamycin, spermine tetrahydrochloride, and heterogeneous tRNA from E. coli were supplied by Sigma. l-[2,3,4,5,6-3H]Phenylalanine and [γ-32P]ATP were purchased from Amersham Biosciences. Avian myeloblastosis virus reverse transcriptase was from Roche Diagnostics. dNTPs and dideoxy-NTPs were from Roche Applied Science. ABA-spermine was synthesized and purified according to Clark et al. (26Clark C.T. Swank R.A. Morgan J.E. Basu H. Matthews H.R. Biochemistry. 1991; 30: 4009-4020Crossref PubMed Scopus (18) Google Scholar). Cellulose nitrate filters (type HA; 24-mm diameter, 0.45-μm pore size) were from Millipore Corp. (Bedford, MA). Biochemical Preparations—Salt-washed (0.5 m NH4Cl), polyamine-depleted 70 S ribosomes and partially purified translation factors were prepared from E. coli K12 cells as reported previously (27Amarantos I. Xaplanteri M.A. Choli-Papadopoulou T. Kalpaxis D.L. Biochemistry. 2001; 40: 7641-7650Crossref PubMed Scopus (12) Google Scholar). Before their use, ribosomes were activated in buffer containing 20 mm magnesium acetate and 150 mm NH4Cl by incubation for 20 min at 42 °C. Samples were then cooled to 0 °C, and the Mg2+ concentration was normalized to 4.5 mm. Ac-[3H]Phe-tRNAPhe was prepared from heterogeneous E. coli tRNA, as described by Xaplanteri et al. (28Xaplanteri M.A. Petropoulos A.D. Dinos G.P. Kalpaxis D.L. Nucleic Acids Res. 2005; 33: 2792-2805Crossref PubMed Scopus (43) Google Scholar). It was charged to 77%, 100% being 28 pmol of Phe per A260 unit (Sigma). Post-translocation complex of poly(U)-programmed ribosomes, complex C, bearing tRNAPhe at the E-site and AcPhe[3H]tRNAPhe at the P-site was prepared in buffer A (100 mm Tris/HCl, pH 7.2, 4.5 mm magnesium acetate, 150 mm NH4Cl, and 6 mm 2-mercaptoethanol) and purified as shown in the same study. Whenever desired, 100 μm spermine or 50 μm spermine and 2 mm spermidine were also included in buffer A. In the presence of polyamines, more than 50% of the used ribosomes was converted to complex C. This fraction was almost fully reactive toward puromycin. Photoaffinity Labeling, Mapping of ABA-Spermine Cross-linking Sites in 23 S rRNA, and Chemical Modification—Complex C was photolabeled with 100 μm ABA-spermine in HEPES buffer containing 4.5 mm Mg2+ and 150 mm NH+4, and the photolabeled product was purified as described elsewhere (28Xaplanteri M.A. Petropoulos A.D. Dinos G.P. Kalpaxis D.L. Nucleic Acids Res. 2005; 33: 2792-2805Crossref PubMed Scopus (43) Google Scholar). The sites in 23 S rRNA to which ABA-spermine was crosslinked were identified by primer extension analysis, as described by Stern et al. (29Stern S. Moazed D. Noller H.F. Methods Enzymol. 1988; 164: 481-489Crossref PubMed Scopus (400) Google Scholar). Binding of Ac[3H]Phe-tRNA to the P and A sites of Poly(U)-programmed Ribosomes—70 S ribosomes programmed with poly(U) were incubated for 30 min at 25 °C in buffer A containing 0.4 mm GTP and uncharged tRNAPhe at a molar ratio to ribosomes 1.5:1 to pre-fill the P-site. Subsequently, Ac[3H]Phe-tRNA was added and incubated for up to 30 min at 25 °C to allow A-site binding. Whenever required, 30 μm clindamycin was included in the binding buffer. The level of the A-site bound Ac[3H]Phe-tRNA was measured by nitrocellulose filtration. Bound at this site, Ac[3H]Phe-tRNA was almost no reactive toward puromycin. Total binding was measured by using poly(U)-programmed 70 S ribosomes with no prefilled P-sites. The P-site bound Ac[3H]Phe-tRNA was estimated from the total binding by titration with puromycin (2 mm, 2 min at 25 °C). Kinetics of the Puromycin Reaction—The reaction between untreated or photolabeled complex C and puromycin (S) (Scheme 1) was performed at 5 or 25 °C in buffer A containing 5nm ribosomes in the form of complex C and puromycin at concentrations varying from 0.1 to 2 mm (30Petropoulos A.D. Xaplanteri M.A. Dinos G.P. Wilson D.N. Kalpaxis D.L. J. Biol. Chem. 2004; 279: 26518-26525Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). Whenever required, 100 μm spermine or 50 μm spermine and 2 mm spermidine were also included in the reaction mixture. The product (P), Ac[3H]Phe-puromycin, was extracted in ethyl acetate, and its radioactivity was measured in a liquid scintillation spectrometer. Background controls (minus puromycin) were subtracted. The product was expressed as percentage (x) of the radioactivity contained in complex C. The values of x, appropriately corrected (30Petropoulos A.D. Xaplanteri M.A. Dinos G.P. Wilson D.N. Kalpaxis D.L. J. Biol. Chem. 2004; 279: 26518-26525Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), were fitted into Equation 1,ln100100-x=kobs·t(Eq. 1) where t is the reaction time, and kobs is the pseudo-first-order rate constant. As previously justified (31Synetos D. Coutsogeorgopoulos C. Biochim. Biophys. Acta. 1987; 923: 275-285Crossref PubMed Scopus (38) Google Scholar, 32Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar), the kobs value is related to the puromycin concentration by Equation 2,kobs=k3[S]Ks+[S](Eq. 2) where k3 represents the catalytic rate constant of PTase, and Ks is the dissociation constant of the encounter complex between puromycin and complex C. The k3 and Ks values were calculated from DR plots (1/kobs versus 1/[S]) derived from Equation 2. Kinetics of the Puromycin Reaction Inhibition by Clindamycin—In this series of experiments, complex C in buffer A was added to a mixture containing puromycin and clindamycin. At 25 °C, biphasic time-plots (ln[100/(100 - x)] versus t) were obtained. The initial slope of each curve was taken as a measure of the apparent rate constant (kobs)o at the early phase of the reaction. Similarly, the slope of the slow-onset straight line was taken as the value of the apparent rate constant (kobs)s at the late phase of the reaction. Alternatively, the (kobs)o and (kobs)s values were estimated by fitting the data to a two-exponential function. Both methods of data processing resulted in the same rateconstant values. At 5 °C, single-phase time plots were obtained, and one value for kobs was calculated. Statistics—One-way of variance was used to estimate the mean value and data variability. Statistically significant differences were determined using the unpaired Student's t test. Effect of Clindamycin on the Binding of Ac[3H]Phe-tRNA to the A and P sites of Poly(U)-programmed Ribosomes—Binding experiments were performed at 25 °C under several ionic conditions in the presence or in the absence of clindamycin. To estimate the maximum level of binding, we followed the entire course of the reaction. As shown in Fig. 2, clindamycin hardly affects the binding of AcPhe-tRNA to both sites of poly(U)-programmed ribosomes if the binding is performed in the absence of polyamines; although a small increase in binding is visible in the presence of clindamycin, the differences from the control values are not statistically significant (p < 0.05). In accordance with previous results (24Bartetzko A. Nierhaus K.H. Methods Enzymol. 1988; 164: 650-658Crossref PubMed Scopus (67) Google Scholar, 33Karahalios P. Mamos P. Karigiannis G. Kalpaxis D.L. Eur. J. Biochem. 1998; 258: 437-444Crossref PubMed Scopus (5) Google Scholar), spermine or a mixture of spermine and spermidine stimulates the AcPhe-tRNA binding and enhances the stability of the formed 70 S initiation complex. The addition of clindamycin into the polyamine buffer improves further the AcPhe-tRNA binding to both sites but impairs the stability of the initiation ribosomal complex formed. Inhibition of AcPhe-Puromycin Synthesis by Clindamycin—The reaction shown in Scheme 1 represents the synthesis of AcPhe-puromycin from complex C and puromycin in excess. It proceeds as an irreversible pseudo-first-order reaction in which C is converted to C′, a species of complex C stripped of Ac[3H]Phe-tRNA and, thus, unable to react with puromycin for a second cycle. The integrated kinetic law expressed by Equation 1 predicts that the progress curve of AcPhe-puromycin synthesis is expressed by a straight line at each concentration of puromycin. The predicted linearity is indeed observed experimentally. A representative plot obtained at 200 μm puromycin and 25 °C is shown in Fig. 3A, upper line. However, when complex C reacts with a mixture of puromycin and clindamycin at 25 °C, the reaction displays biphasic kinetics, characterized by an initial phase followed by a late phase (Fig. 3A, lower line). The deviation from linearity is of vital importance in our analysis because it suggests a delay in the onset of inhibition. Analysis of the initial slopes by DR plotting and slope replotting (Figs. 3, B and C) leads to the identification of an initial phase of simple competitive inhibition, with one molecule of clindamycin participating in the mechanism of inhibition. This type of inhibition survives over a narrow range of inhibitor concentrations ([I] < 10 μm). The Κi value of 5.5 μm found at 4.5 mm Mg2+ and 150 mm NH+4 agrees well with the value of 8 μm for clindamycin affinity measured by footprinting analysis (4Douthwaite S. Nucleic Acids Res. 1992; 20: 4717-4720Crossref PubMed Scopus (68) Google Scholar). However, it does not markedly differ from the Κi value for lincomycin so as to justify the superiority of the in vivo clindamycin potency. It should be mentioned that this inhibition constant determines only the initial encounter of clindamycin with complex C, ignoring late events of the drug-ribosome interaction. On the other hand, comparisons between in vivo and in vitro processes should be made cautiously, given that several factors in vivo may render one drug more potent than another. With increasing concentrations of clindamycin the competition at 25 °C becomes stronger (Fig. 3C). We suppose that at high concentrations of clindamycin a new relation of the drug to the puromycin binding site is established. This alteration becomes more pronounced when the late phase of puromycin reaction is analyzed. As shown in Fig. 4A, with increasing concentrations of clindamycin the DR plots meet the negative 1/[S] axis at the same point, whereas the slopes of the lines increase, approaching a plateau (Fig. 4B). Such a behavior characterizes inhibitors of the partial-noncompetitive type. Nevertheless, analysis of the Fig. 4A data by Hill-plotting (not shown) reveals that again, only one molecule of clindamycin participates in the mechanism of inhibition. Combined, these results suggest that clindamycin (I) reacts transiently with complex C to form the encounter complex CI, which is then isomerized slowly to a more stable complex, termed C*I. The partial-noncompetitive inhibition established at the late phase also implies that complex C*I, contrary to complex CI, is capable of accommodating the substrate and producing AcPhe-puromycin albeit with a lower catalytic rate constant than k3. The two phases of inhibition obtained at 25 °C are compatible with a model shown in Scheme 2.SCHEME 2View Large Image Figure ViewerDownload Hi-res image Download (PPT) We assume that at the late phase of the puromycin reaction, the unimolecular change of CI to C*I is at equilibrium. Consequently, k7[C*I] = k6[CI]. This also implies that P is produced via both the k3 and k3* steps. The Ks*, k3*, k6, and k7 values estimated as described previously (Refs. 30Petropoulos A.D. Xaplanteri M.A. Dinos G.P. Wilson D.N. Kalpaxis D.L. J. Biol. Chem. 2004; 279: 26518-26525Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar and 34Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1993: 39-43Google Scholar; see also supplemental data 3Derivation of the kinetic equations used in this report and data processing for estimation of the Ks*, k3*, k6, and k7 values are provided as supplemental data. are presented in Table 1. According to these values, the overall association constant at 4.5 mm Mg2+ and 150 mm NH+4, kassoc, concerning both steps of the clindamycin interaction with complex C, is equal to 3.81 μm−1 min−1. As a consequence, the overall dissociation constant, k7/kassoc, becomes 13-fold lower than Ki, a fact suggesting that the affinity of complex C for the drug is much higher than that expressed just by the Ki alone.TABLE 1Equilibrium and kinetic constants derived from analysis of the inhibition of AcPhe-puromycin synthesis by clindamycinPuromycin reaction conditionsConstant (unit)4.5 mm Mg2+, 150 mm NH+44.5 mm Mg2+, 150 mm NH+4, 100 μm spermine4.5 mm Mg2+, 150 mm NH+4, 50 μm spermine, 2 mm spermidine4.5 mm Mg2+, 150 mm NH+4, complex C photolabeled by ABA-sperminek3 (min−1)2.00 ± 0.022.86 ± 0.05*2.50 ± 0.05*2.63 ± 0.05*KS (μm)486 ± 28510 ± 22505 ± 25496 ± 24Ki (μm)5.50 ± 0.301.04 ± 0.06*1.29 ± 0.07*1.50 ± 0.09*K6 (min−1)19.40 ± 1.2010.20 ± 0.70*10.26 ± 0.60*8.70 ± 0.60*k7 (min−1)1.55 ± 0.121.46 ± 0.101.34 ± 0.081.21 ± 0.09*k3* (min−1)0.049 ± 0.0220.052 ± 0.0280.044 ± 0.0270.049 ± 0.028Ks* (μm)404 ± 90445 ± 95403 ± 87412 ± 90kassoc (μm−1 min−1)3.81 ± 1.2011.20 ± 0.90*9.00 ± 0.77*6.60 ± 0.72*k7/kassoc (μm)0.41 ± 0.130.13 ± 0.01*0.15 ± 0.01*0.18 ± 0.02* Open table in a new tab In contrast to the results seen at 25 °C, single-phase time plots were obtained at 5 °C. A representative plot obtained at 200 μm puromycin and 1 μm clindamycin is shown in Fig. 3A (midline). As revealed by detailed kinetic analysis, increasing the concentration of the drug does not alter the type of inhibition, which remains simple-competitive (Ki = 5.6 μm) throughout the time course of the reaction. Polyamines Enhance the Inhibitory Effect of Clindamycin—To reveal the effect of spermine on the clindamycin potency, we re-analyzed the mechanism of inhibition using complex C, which was prepared in buffer A containing 100 μm spermine and then interacted at 25 °C with puromycin in the same buffer. In agreement with previous results obtained at 6 mm Mg2+ (30Petropoulos A.D. Xaplanteri M.A. Dinos G.P. Wilson D.N. Kalpaxis D.L. J. Biol. Chem. 2004; 279: 26518-26525Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, 33Karahalios P. Mamos P. Karigiannis G. Kalpaxis D.L. Eur. J. Biochem. 1998; 258: 437-444Crossref PubMed Scopus (5) Google Scholar), the addition of spermine at this concentration increases the k3 value of PTase without affecting the Ks dissociation constant (Table 1). Consequently, the ratio k3/Ks expressing the activity status of PTase is enhanced by 43%. It should be noticed here that the k3 values estimated in the present study agree with previous literature values (31Synetos D. Coutsogeorgopoulos C. Biochim. Biophys. Acta. 1987; 923: 275-285Crossref PubMed Scopus (38) Google Scholar, 36Polacek N. Swaney S. Shinabarger D. Mankin A.S. Biochemistry. 2002; 41: 11602-11610Crossref PubMed Scopus (16) Google Scholar, 37Blanchard S.C. Harold D.K. Gonzalez Jr R.L. Puglisi J.D. Chu S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12893-12898Crossref PubMed Scopus (373) Google Scholar), but they are lower than those measured by fast kinetics (32Katunin V.I. Muth G.W. Strobel S.A. Wintermeyer W. Rodnina M.V. Mol. Cell. 2002; 10: 339-346Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). This discrepancy seems to be due to the different experimental conditions and substrates used and, more probably, to a different accommodation of puromycin into the catalytic center. Nevertheless, the thermodynamic behavior of both systems is similar (38Dinos G. Kalpaxis D.L. Wilson D.N. Nierhaus K.H. Nucleic Acids Res. 2005; 33: 5291-5296Crossref PubMed Scopus (35) Google Scholar). Despite the stimulatory effect on PTase, spermine does not change the type of inhibition by clindamycin. Thus, clindamycin again inhibits peptide-bond formation at 25 °C by binding initially to complex C in competition with puromycin. Subsequently, a slow isomerization occurs, resulting in a tighter complex C*I that accepts puromycin but produces AcPhe-puromycin with a much lower catalytic rate constant. Nevertheless, the values of certain kinetic parameters differ from those obtained in the absence of spermine (Table 1). Namely, the Ki value becomes 5-fold smaller. In addition, the k6 value is reduced by 52%, whereas the k7 value remains essentially constant. As a consequence, the overall association rate constant, (k6 + k7)/Ki, becomes three times higher, a fact that favors the formation of complex C*I. Similar changes in the kinetic p" @default.
• W2081482806 created "2016-06-24" @default.
• W2081482806 creator A5022471377 @default.
• W2081482806 creator A5049471117 @default.
• W2081482806 creator A5058297155 @default.
• W2081482806 date "2006-08-01" @default.
• W2081482806 modified "2023-09-27" @default.
• W2081482806 title "Unraveling New Features of Clindamycin Interaction with Functional Ribosomes and Dependence of the Drug Potency on Polyamines" @default.
• W2081482806 cites W1486369891 @default.
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