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Peptide antibiotics are known to contain non-protein amino acids, D-amino acids, hydroxy acids, and other unusual constituents. In addition they may be modified by N-methylation and cyclization reactions. Their biosynthetic origin has been connected in many cases to an enzymatic system referred to as the ‘thiotemplate muttienzymic mechanism’. This mechanism includes the activation of the constituent residues as adenylates on the enzymic template, the acylation of specific template thiol groups, epimerization or N-methylation at this thioester stage, and polymerization in the sequence directed by the multienzymic structure with the aid of 4′-phosphopantetheine as a cofactor, including possible cyclization or terminal modification reactions. The reaction sequences leading to gramicidin S, tyrocidine, cyclosporine, bacitracin, polymyxin, actinomycin, enniatin, beauvericin, δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine and linear gramicidin are discussed. The structures of the multienzymes, their genetic organization, the biological functions of these peptides and results on related systems are discussed. [ABSTRACT FROM AUTHOR]

From rat brain extracts, two carnosine-degrading enzymes have been identified and partially purified by ion-exchange chromatography, hydrophobic interaction chromatography on phenyl-Sepharose CL-4B and gel filtration. These enzymes exhibit distinct differences in their chemical characteristics and substrate specificities. One enzyme, designated carnosinase, preferentially hydrolyzes carnosine and exhibits a low Km value (0.02 mM) towards this substrate. Carnosinase also degrades anserine but not homocarnosine or homoanserine. The other carnosine-degrading enzyme hydrolyzes βAla-Arg considerably faster than carnosine and, therefore, has been tentatively designated βAla-Arg hydrolase. This enzyme exhibits high Km values with carnosine (Km = 25 mM) and βAla-Arg (Km = 2 mM). Homocarnosine and γ-aminobutyryl-arginine are not degraded by βAla-Arg hydrolase. Neither enzyme is inhibited by agents reactive on activated hydroxyl groups, such as diisopropyl fluorophosphate, and also not by a variety of peptidase inhibitors of microbial origin or from other sources. Carnosinase is also not inhibited by bestatin but βAla-Arg hydrolase, although not an aminopeptidase, is strongly inhibited by this aminopeptidase inhibitor (IC50 = 50 nM). While carnosinase is strongly inhibited by thiol-reducing agents such as dithioerythritol and 2-mercaptoethanol, βAla-Arg hydrolase is stabilized and activated by these substances. Both enzymes are strongly inhibited by metal-chelating agents. Carnosinase, however, is not dependent on exogeneously added metal ions and is strongly inhibited by Mn2+ as well as by heavy metal ions. In contrast, βAla-Arg hydrolase requires Mn2+ ions for full enzymatic activity. Based on these differences, selective incubation conditions could be evaluated in order to determine specifically both enzyme activities in crude tissue extracts. In rat, both enzymes are present in all tissues tested, except skeletal muscles, but considerable differences in their relative distribution among different tissues are also observed. [ABSTRACT FROM AUTHOR]

Investigates the substrate specificity of a serum enzyme which degrades thyroliberin into pyroglutamic acid and His-Pro-NH 2 . Rapid degradation of thyroliberin and luliberin; Pyroglutamyl-histidine bond; Hydrolysis.

δ-(L-α-Aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase, the multienzyme catalyzing the formation of ACV from the constituent amino acids and ATP in the presence of Mg2+ and dithioerythritol, was purified about 2700-fold from Streptomyces clavuligerus. The molecular mass of the native enzyme as determined by gel filtration chromatography is 560 kDa, while that determined by denaturing gel electrophoresis is 500 kDa. The enzyme is able to catalyze pyrophosphate exchange in dependence on L-cysteine and L-valine, but no L-α-aminoadipic-acid-dependent ATP/PP1 exchange could be detected. Other L-cysteine- and L-valine-activating enzymes present in crude extracts were identified as aminoacyl-tRNA synthetases which could be separated from ACV synthetase. The molecular mass of these enzymes is 140 kDa for L-valine ligase and 50 kDa for L-cysteine ligase. The dissociation constants have been estimated, assuming three independent activation sites, to be 1.25 mM and 1.5 mM for cysteine and ATP, and 2.4 mM and 0.25 mM for valine and ATP, respectively. The enzyme forms a thioester with α-aminoadipic acid and with valine in a molar ratio of 0.6:1 (amino acid/enzyme). Thus, the bacterial ACV synthetase is a multifunctional peptide synthetase, differing from fungal ACV synthetases in its mechanism of activation of the non-protein amino acid. [ABSTRACT FROM AUTHOR]