Hydroxamic acids formation from peptides

On this basis the extent to which an enzyme-catalyzed transamidation will occur will depend on the relative concentration of water and the replacement reagent, and the relative affinity of the replacement agent for the ES-complex. Durell and Fruton (51) have studied papain-catalyzed hydroxamic acid formation from o-benzoyl-L-argininamide. Their calculations show that hydroxylamine is about 420 times more efficient in its reaction with the enzyme-substrate complex than is water. Preliminary observations suggest that when amino acid amides or peptides are the attacking molecule the efficiency is even greater. Moreover, papain is a much more effective catalyst for transamidation than trypsin. 

Many investigators have studied the incorporation of labeled amino acids into proteins. The system involved in mammalian cells seems to be associated with microsomes, but requires factors from the soluble portion of the cell. Both ATP and GTP are required. The function of the ATP may be to form activated amino acids. Several systems have been found with relative specificity for various amino acids that catalyze the exchange of pyrophosphate with ATP in the presence of the amino acid, and presumably form adenyl-amino acid compounds. The activated amino acids react with hydroxylamine to form hydroxamic acids. Several reactions have been considered as models for peptide bond formation. These include the formation of hippuric acid, in which benzoyl CoA condenses with glycine in a reaction similar to the acetylation of... 

O, D) A hypothetical pathway is the formation of an acylimino acid unit by N-hydroxylation of the amino acid within the peptide chain followed by loss of water. a-Acylimino carboxylic acid derivatives readily rearrange to acylenamino carboxylic acid derivatives 314). N-Hydroxy compounds, such as mycelianamide and pulcherrimic acid [for a review on cyclic hydroxamic acids see ref. (26)], have been found within the class of dehydrocyclopeptides. Although N-hydroxypyra-zinones are common, it is not known whether these compounds are biogenetically linked with the piperazinediones. It is appropriate to point out that N-hydroxylation of amides is a familiar degradative pathway in the metabolic processes of higher organisms 412). The facile elimination of water from N-acylhydroxylamino acids to form acylenamino acids 377) should also be noted in this context. 

It was expected that if amino acids were used instead of hydroxylamine, peptides would be formed in the experiments of aminoacyl hydroxamate formation 31). However, when glycine is warmed with ATP and MgCl2 in the solid state (derived from the solution), peptide is not formed but adenylyl-(5 -> N)-glycine (gly-N-pA) is formed34> 35). When imidazole is used instead of amino acids, adenosine 5 -phosphorimidazolide (ImpA) is formed in a few percent yield 34). 

Application and Principle This procedure is used to determine transglutaminase activity in preparations derived from Streptoverticillium mobaraense var. The assay is based on the enzymatic formation of a glutamic acid y-hydroxamate in a glutaminyl residue in the substrate peptide with another substrate, hydroxylamine. The amount of the glutamic acid y-hydroxamate formed as a red complex with ferric ion in acidic conditions at 37° is measured spectrophotometrically. 

---