Penicillin biosynthesis: energy requirement for tripeptide precursor formation by delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase from Acremonium chrysogenum.
In nonribosomal peptide formation by multifunctional enzymes, peptide synthetases catalyze the activation and directed condensation of amino acids. The peptide synthetase involved in penicillin biosynthesis (ACV synthetase) forms the tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine from the respective L-amino acids and ATP. So far, the energy requirements for the nonribosomal process have not been clearly established. For ACV synthetase we show that ATP consumption depends on the reaction conditions employed. By simultaneously estimating peptide and AMP production by employing fluorescence detection and UV spectroscopy, respectively, we have determined the energy consumption with high accuracy. Under unfavorable reaction conditions more than 20 mol of ATP are consumed/mol of tripeptide formed, while optimal conditions permit the expected energy requirement of one ATP for each carboxyl group activation, corresponding to three ATP for tripeptide formation. The third ATP is required for the activation of L-valine to maintain the valyl-thioester stage for epimerization and peptide bond formation, and this high-energy bond is sacrificed by hydrolytic removal of the product. No extra energy is required for the directed transport in peptide elongation. Additional energy consumed has been traced to hydrolytic loss of activated intermediates, as has been shown by the analysis of incomplete reaction mixtures.
Kallow
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W
Institute of Biochemistry and Molecular Biology, Technical University Berlin, Germany.
von Döhren
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H
Kleinkauf
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D000164:Acremonium / Q000201:enzymology*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004229:Dithiothreitol / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D009842:Oligopeptides / Q000096:biosynthesis* / Q000378:metabolism; D010406:Penicillins / Q000096:biosynthesis*; D010453:Peptide Synthases / Q000378:metabolism*; D019163:Reducing Agents / Q000494:pharmacology
D000596:Amino Acids; D009842:Oligopeptides; D010406:Penicillins; D019163:Reducing Agents; C011638:5-(2-aminoadipyl)cysteinylvaline; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C095368:alpha-aminoadipyl-cysteinyl-valine synthetase; D004229:Dithiothreitol