title: Nonribosomal peptide synthetases-evidence for a second ATP-binding site.
abstract: delta-(L-alpha-Aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS) catalyses, via the protein thiotemplate mechanism, the nonribosomal biosynthesis of the penicillin and cephalosporin precursor tripeptide delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine (ACV). The complete and fully saturated biosynthetic system approaches maximum rate of product generation with increasing ATP concentration. Nonproductive adenylation of ACVS, monitored utilising the ATP-[32P]PP(i) exchange reaction, has revealed substrate inhibition with ATP. The kinetic inhibition pattern provides evidence for the existence of a second nucleotide-binding site with possible implication in the regulatory mechanism. Under suboptimal reaction conditions, in the presence of MgATP(2-), L-Cys and inorganic pyrophosphatase, ACVS forms adenosine(5')tetraphospho(5')adenosine (Ap(4)A) from the reverse reaction of adenylate formation involving a second ATP molecule. The potential location of the second ATP binding site was deduced from sequence comparisons and molecular visualisation in conjunction to data obtained from biochemical analysis.
authors.affiliation: Max-Volmer-Institut für Biophysikalische Chemie und Biochemie, Technische Universität Berlin, Franklinstrasse 29, 10587 Berlin, Germany.
authors.forename: Wibke
authors.initials: W
authors.lastname: Kallow
authors.forename: Maja
authors.initials: M
authors.lastname: Pavela-Vrancic
authors.forename: Ralf
authors.initials: R
authors.lastname: Dieckmann
authors.forename: Hans
authors.initials: H
authors.lastname: von Döhren
mesh_terms: D000164:Acremonium / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002511:Cephalosporins / Q000096:biosynthesis; D007700:Kinetics; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry; D010406:Penicillins / Q000096:biosynthesis; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation
chemical_list: D002511:Cephalosporins; D009842:Oligopeptides; D010406:Penicillins; D000255:Adenosine Triphosphate; D010453:Peptide Synthases