11.10.15 - Lecture 15- Antibiotics and RNA catalysts R Penicillin antibiotics-inactivated by beta-lactamase's ability to perform a fast deacylation of the inhibitor-enzyme adduct S R N O O CO2 R H2O S O HN O CO2 Enz-Ser OH Enz-Ser Inhibitors of beta-lactamases R Enz-Lys NH2 O R O HN O R SO2- Enz-Ser - O CO2 OH + O Enz-Ser NH O O2S O H2N CO2 - Enz-Ser Stable enzyme adduct builds up O O S HN OH R O Enz-Ser R Lys-Enz NH HN O CO2 Enz-Ser CO2 Enz-Lys O O S R N O CO2 OH Enz-Ser O O S HN OH S Deacylation CO2 Using an alternative leaving group- R O O N O R OH CO2 CO2 OH O R OH + HN O HN O Enz-Ser O O CO2 Enz-Ser In general,beta-lactam antibiotics are not affected by drug pumps as their target resides in the periplasmic space Targeting D-Ala D-Ala ligase O ONH2 O O O O P O P O O- AMP O+ - O O O P O- NH2 O H2N - + H2N H2N P O H2N O O- O - O - O- H N O H2N Design of an inhibitor of alanine ligase -inhibition is dependent on presence of ATP - off rate t1/2 ~17 days - koffINH << koff PDT, 108-109 slower off rate -example of slow binding/slow off rate inhibitor OO P OO O O H2N CO2Enzyme performs slow transphosphorylation in presence of ATP O CO2- P O O P OO- Better TS analog Previously discussed antibiotic mechanisms targeting D-Ala D-Ala -transpeptidase inhibitor -inhibitor of alanine racemase -inhition of D-Ala ligase Vancomycin-a natural product 'receptor' which specifically binds to D-Ala-D-Ala via a network of H-bonding and blocks transpeptidase Vancomycin Resistance -bacteria use lactic acid instead of D-Ala -ester linkage is much less favorable, repulsive interactions between oxygen lone pairs, loss of H-bond O D-Ala O O O O- NH2 O- OOH Lactic acid Loss of H-bond and introduction of unfavorable interaction RNA catalysts -RNA can carry out catalysis -most often dependent on presence of Magnesium Self splicing RNA in tetrahymena General reaction3' YYUpN + G-OH3' G-OH YYU-OH3' + GpN (Y = pyrimidine base) 5' Intron splicing cucucu * AGGGAGG 5' U U U G*uaaggu-OH 3' cucucu-OH 3' AGGGAGG-UUU*G-OH3' 5' cucucu*uaagu-3' G*uaaggu-OH 3' (ligated exons) + 3' HO-G O-G 5' G*UUU-OH 5' G*UUU AGGGAGG AGGGAGG* CIVS Catalytic RNA -in order to be catalytic, the catalyst molecule must remain unchanged at the end of the reaction 3' HO-G RNA which ligates to DNA substrateRNA ligaseACUCG-OH 3' ACUCG-OH 3' CCCTCCTpA3TA3TA CCCUCCUpA3UA3UA GGGAGG-5' GGGAGG-5' CCCCpC GGGAGG-5' C4 3' HO-CpG CCCCC-OH GGGAGG-5' C6 ACUCGpA3TA3TA ACUCGpA3UA3UA S GGGAGG-5' primer sequence enzyme can be amplified selectively since only catalytic RNAs are selected for GGGAGG-5' Amplification of starting RNA with RT rxn, PCR, and in vitro transcption with T7 polymerase. Sequence diversity generated by error prone PCR conditions or using an error prone DNA polymerase 3' HO-G RNAs with increased catalytic activities GGGAGG-5' Starting RNA kcat - 2x10-4 min-1 Km ~7 M After selection kcat - 0.007 min-1 Km ~2 M + C4 + C6 (catalytic RNA unchanged) (catalyzes disproportionation reaction) RNA RT DNA PCR DNA In vitro transcription RNA T7 Promoter reverse transcriptase DNA polymerase (error prone) (amplification step) T7 polymerase (amplification step) An RNA ligase selected from randomized sequence -randomize 220 bases -library will be incomplete (4220 potential members possible); must assume many potential solutions Using this strategyan RNA enzyme was discovered with a t1/2 of 5 minutes vs. 33 years compared to the uncatalyzed reaction (107 rate acceleration)
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