Abstract
Actinomycin Synthetase II (ACMS II), which activates threonine and valine by a thioltemplate mechanism during the synthesis of the actinomycin half-molecule 4-methyl-3-hydroxyanthranilic acid (4-MHA) pentapeptide lactone, was purified to near homogeneity from Streptomyces chrysomallus. It is a single polypeptide chain of M(r) 280,000 and contains 4'-phosphopantetheine as a covalently bound prosthetic group. ACMS II charges itself with threonine but not with the 4-MHA analogue p-toluic acid via a specific sulfhydryl group at the expense of ATP. Charging of ACMS II with p-toluic acid in thioester linkage took place, however, only when actinomycin synthetase I (ACMS I), a 4-MHA-AMP ligase, was present. In the additional presence of L-threonine, enzyme-bound p-toluyl-L-threonine was formed on ACMS II. The latter compound was also formed when chemically synthesized p-toluic acid adenylate was added instead of ACMS I and p-toluic acid. This indicates that p-toluic acid adenylate is a free intermediate in the reaction and that charging of the enzyme and acylation of threonine are both catalyzed by ACMS II rather than by ACMS I. Chemically synthesized thioesters of p-toluic acid and coenzyme A, pantetheine, or beta-alanyl-cysteamine reacted with ACMS II, threonine, and ATP with formation of enzyme-bound p-toluyl-threonine. In contrast, p-toluyl-cysteamine thioester was inactive, which indicates structural constraints in the reactivity of free thioesters of p-toluic acid with ACMS II. Such constraints obviously require structural similarity of the artificial substrate to a p-toluic acid thioester formed on the enzyme's surface in the course of the reaction. Since free coenzyme A was not involved in the charging of p-toluic acid or in p-toluyl-threonine formation, the sulfhydryl group of the 4'-phosphopantetheine cofactor is most likely the primary acceptor of p-toluic acid (or 4-MHA) in the initiation of peptide lactone formation.
Highlights
Actinomycin Synthetase I1 (ACMS 11),which acti- onepentapeptideringwith4-methyl-3-hydroxyanthranilic vates threonine and valineby a thioltemplate mecha- acid (4-MHA)’ at the NHz terminus (Fig. 1).Actinomycin nism duringthesynthesis of the actinomycinhalf- synthetases I1 and I11 which serve as protein thioltemplates molecule 4-methyl-3-hydroxyanthranilaicid
ACMS I1 charges itself with threonine but not with the 4-MHA analogue p-toluic acid via a specific sulfhydrylgroup at the expense of ATP
Since free coenzyme A was not to threonine in the peptide chain of 4-MHApentapeptide involved in the charginogf p-toluic acidor in p-toluyl- lactone
Summary
From the Institut fur Biochemie und MolekularBeiologie, Technisck Uniuersitat Berlin, Franklinstrasse, D - W-1000Berlin 10, Germany. Since free coenzyme A was not to threonine in the peptide chain of 4-MHApentapeptide involved in the charginogf p-toluic acidor in p-toluyl- lactone This suggests that ACMS I and I1 must interact in threonine formation, the sulfhydryl group of the 4‘- some way to initiate acylpeptide lactone synthesis. In this phosphopantetheine cofactoris most likely the primary acceptor of p-toluic acid (or 4-MHA) in the initiation of peptide lactone formation. Paper we report the purification of ACMS I1 and the way in which it interacts with ACMS I, the 4-MHA activating enzyme It wasshown, that ACMS I1 charges itself with the aromatic carboxylic acid in thioester linkage when ACMS I ispresent.ThisenablesACMS.
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