Frank B. Armstrong

Formylated peptides from cyanogen bromide digests identified by fast atom bombardment mass spectrometry

Exposure of proteins to 70% formic acid during cyanogen bromide digestion can result in formation of artifact peaks during subsequent purification by HPLC and detection of [M + H + 28]+ ions during analysis by fast atom bombardment (FAB) mass spectrometry. Following cyanogen bromide digestion, peptides from equine heart cytochrome c, bacteriorhodopsin, bovine adrenal medulla dodecapeptide, and bovine adrenal peptide E were analyzed by positive ion FAB mass spectrometry. The cyanogen bromide peptides of cytochrome c and bacteriorhodopsin showed mixtures of formylated ions, [M + H + 28]+, and nonformylated ions, [M + H]+. Bovine adrenal medulla dodecapeptide and bovine adrenal peptide E were not formylated during digestion. Formylated peptides could be resolved from the corresponding nonformylated peptides using reversed-phase HPLC. Instability of the formylated peptides prevented localization of the adduct by Edman degradation. However, B/E-linked scanning during FAB mass spectrometric analysis with collisional activation of the [M + H + 28]+ ion of a cyanogen bromide peptide from cytochrome c suggested that formylation occurred at a threonine residue. On the basis of stability measurements in aqueous solution and analysis by FAB mass spectrometry, it was determined that serine and threonine residues are the most likely sites of esterification by formic acid during cyanogen bromide digestion of proteins. Furthermore, substitution of 70% trifluoroacetic acid for formic acid during cyanogen bromide digestion eliminated formylation and generated little or no trifluoroacetylation.

Branched-chain amino-acid aminotransferase of Salmonella typhimurium. I. Crystallization and preliminary characterization☆

Abstract 1. 1. A protocol for the purification of transaminase B (branched-chain amino-acid:2-oxoglutarate aminotransferase, EC 2.6.1.6) from Salmonella typhimurium that includes the following steps: (a) heat treatment; (b) ammonium sulfate fractionation; and (c) DEAE-cellulose column chromatography is presented. 2. 2. Crystallization of the enzyme is accomplished by an ammonium sulfate treatment of the purified preparation. 3. 3. Results of disc gel electrophoresis provide evidence that the crystalline enzyme is highly purified and free of any significant contamination. 4. 4. Basic enzymatic and spectral characteristics of the crystalline enzyme are presented.

Stereoselectivity and stereospecificity of the α,β-dihydroxyacid dehydratase and from Salmonella typhimurium

Abstract 1. 1. In addition to the known 2R,3R- and 2R, 3S-2,3-dihydroxy-3-methylpentanoic acids (DHI), the 2S,3S- and 2S,3R-isomers were prepared. 2S-2,3-Dihydroxy-3-methylbutanoic acid (DHV) was also prepared in addition to the known 2R-isomer. 2. 2. The six dihydroxy acids were examined for their ability to promote the growth of isoleucine-valine ( ilv )-requiring strains of Salmonella typhimurium and to serve as substrates fro the α,β-dihydroxyacid dehydratase of the same organism. 3. 3. Only 2R,3R-2,3-dihydroxy-3-methylpentanoic and 2R-2,3-dihydroxy-3-methylbutanoic acids supported growth of the ilv strains of S. typhimurium . 4. 4. α,β-Dihydroxyacid dehydratase utilized the three isomers with the 2R-configuration as substrates but not those with the 2S-configuration. 5. 5. In an additional growth study that utilized the 3R- and 3S-isomers of 3-methyl-2-oxopentanoic acid, the α-keto acid analogue of isoleucine, only the 3S-isomer supported growth. 6. 6. It is concluded that the mechanism of action of the dehydratase is stereospecific in that the proton that is attached to C-3 of the substrate occupies the same stereochemical position as the departing hydroxyl group (Fig. 6).

Stereochemistry of the reductoisomerase and αβ-dihydroxyacid dehydratase-catalysed steps in valine and isoleucine biosynthesis. Observation of a novel tertiary ketol rearrangement

The reductoisomerase of Salmonella typhimurium has a requirement for the 2S-isomer of acetolactate (III, R = Me) while the αβ-dihydroxy-acid dehydratase has a requirement for the 2R configuration but is not stereoselective with respect to the configuration at C-3.

Biosynthesis of valine and isoleucine: synthesis and biological activity of (2S)-α-acetolactic acid (2-hydroxy-2-methyl-3-oxobutanoic acid), and (2R)- and (2S)-α-acetohydroxybutyric acid (2-ethyl-2-hydroxy-3-oxobutanoic acid)

Stereoisomers of the two substrates for the enzyme reductoisomerase of the valine-isoleucine pathway of biosynthesis have been synthesised in optically pure form. These compounds are (2S)-α-acetolactate (2-hydroxy-2-methyl-3-oxobutanoate) and (2R)- and (2S)-α-acetohydroxybutyrate (2-ethyl-2-hydroxy-3-oxobutanoate). The synthesis of (2S)-α-acetolactate represents the first synthesis in optically pure form of the substrate of the enzyme in the valine pathway. Only the (2S)-isomers of these compounds acted as substrates for the reductoisomerase of Salmonella typhimurium.

Structure–activity studies with the αβ-dihydroxyacid dehydratase of Salmonella typhimurium

(2RS,3RS)- and (2RS,3SR)-2,3-Dihydroxybutanoic acids, (2R,3R)-2,3-dihydroxy-3-methypentanoic acid, (2RS)-2-ethyl-2,3-dihydroxypentanoic acid, (2RS,3RS)- and (2RS,3SR)-2,3-dihydroxy-3-methylhexanoic acids, and (2RS,3RS)- and (2RS,3SR)-2,3-dihydroxy-3-methylheptanoic acids were synthesised. These acids, as well as (RS)-2,3-dihydroxy-3-methylbutanoic acid and (RS)-glyceric acid were tested as substrates for the αβ-dihydroxyacid dehydratase of the isoteucine–valine biosynthetic pathway of Salmonella typhimurium. For acids having a propyl group at C-3, the activities were greatly reduced compared with those obtained for the natural substrates (2R,3R)-2,3-dihydroxy-3-methylpentanoic acid [(2R,3R)-DHI] and (R)-2,3-dihydroxy-3-methylbutanoic acid [(R)-DHV]. For acids having an n-butyl substituent at C-3, the activities were close to zero. (2RS,3SR)-2,3-Dihydroxybutanoic acid (threo-isomer) underwent dehydration at a rate comparable with that of (2R,3R)-DHI, the natural substrate in the isoteucine pathway, whereas the (2RS,3RS)-acid (erythro-isomer) had much lower activity and (RS)-glyceric acid had even less activity. These results illustrate differences in the alkyl group requirements with respect to the areas of the binding site of the enzyme that accommodate the C-3 substituents. They also demonstrate the size limits of the alkyl groups that can be accommodated in substrate. analogues.

Biosynthesis of L-valine in Salmonella typhimurium: origin of the diastereotopic methyl groups

When [1,3,5-13C3]-α-acetolactate {[1,3,5-13C3]-2-hydroxy-2-methyl-3-oxobutanoate}(1′) was incubated with a cell-free system from Salmonella typhimurium, the valine (5′) produced was labelled in the C-4 pro-S position, proving that during the tertiary ketol rearrangement catalysed by the enzyme reductoisomerase, the methyl group transfer is to the re face of the trigonal centre at C-3 of α-acetolactate (1′).