Gramicidin S synthetase. Temperature dependence and thermodynamic parameters of substrate amino acid activation reactions

Abstract

In the biosynthesis of the cyclic decapeptide antibiotic gramicidin S, the constituent amino acids are activated by a two-step mechanism involving aminoacyl adenylate and thio ester formation which are both reversible processes. The dissociation constants (KD) for the gramicidin S synthetase-substrate amino acid-thio ester complexes are 100-1000-fold lower compared to the KM data of the preceding aminoacyl adenylate reactions. The affinity for these substrates is appreciably higher at the thio template sites than at the aminoacyl adenylate reaction centers. Therefore, the activation equilibria are quantitatively shifted toward thio ester formation. A set of thermodynamic parameters for the activation processes was determined from the temperature dependence of the KM and KD data. Reaction enthalpies were obtained from a van't Hoff analysis of these constants. delta G degree for the substrate activation reactions of the heavy enzyme of gramicidin S synthetase (GS 2) is predominantly controlled by entropy contributions. In contrast, the overall activation and concomitant racemization of phenylalanine by phenylalanine racemase (GS 1) are exothermic processes which are distinguished by a small negative reaction entropy.

Formation of D‐Phe‐Pro‐Val‐cyclo‐Orn by gramicidin S synthetase in the absence of L‐leucine

“…The specific activities of both enzymes range over 20 -35 nmol gramicidin S min-' (mg protein)-'. These data are 3-4-fold higher than those reported in [7].…”

“…Both components of gramicidin S synthetase were purified as published previously [7]. This procedure was improved efficiently by the introduction of a fast protein liquid chromatography technique (Fig.…”

“…These proteins differ slightly from GS 2 regarding their charge and sedimentation coefficients. The time-consuming sucrose gradient centrifugation step [7] can now be replaced by a few fast runs on Mono Q with even better resolution of GS 2. In this way the time needed for the preparation of GS 2 could be reduced considerably.…”

Physiological and biochemical characteristics of poly γ‐glutamate synthetase complex of Bacillus subtilis

“…Gardner & Troy [6] suggested the occurrence of an extremely unstable PGA synthetase complex in B. licheniformis ATCC9945a. Their proposed reaction mechanism [34] was similar to those of multienzyme systems [35], such as the gramicidin S synthetase complex, which were accompanied by the cleavage of ATP into AMP. It is important for the elucidation of the mechanism of PGA synthesis by the PgsBCA system to know whether ADP or AMP is formed by the ATPase reaction.…”

Editing of non-cognate aminoacyl adenylates by peptide synthetases

“…It has been suggested that thioacylation of peptide synthetases must be faster than hydrolysis, or that it regulates the conformational transition, leading to a faster transfer [18]. The affinity for the substrate amino acid is much higher at the thio template than in the adenylation centres ; consequently the activation equilibria are quantitatively shifted towards thioester formation [36,37]. The significance of a putative editing mechanism involving the thiolation site remains to be investigated further using the respective holo-enzymes.…”