A Reactive, Surface Cysteine Residue of the Class‐II Fructose‐1,6‐Bisphosphate Aldolase of Escherichia coli Revealed by Electrospray Ionisation Mass Spectrometry

Abstract

The state of post-translational modification of the class-I1 fructose-I ,6-bisphosphate aldolase (FBPaldolase) purified from Escherichia coli was examined by electrospray ionisation mass spectrometry (ESI-MS). The mass was larger than that expected from the known DNA sequence by approximately 80 ? 6 Da, suggesting the presence of a covalent modification on the protein. Phosphorylation (+80 Da), a known modification in an FBP-aldolase from Bacillus subtilis and a suspected modification in this E. coli aldolase, was ruled out as the extra mass was readily removed by treatment with dithiothreitol. Purification of aldolase by a protocol which omitted 2-mercaptoethanol from all buffers resulted in the purified protein having the expected mass (39016 Da). The extra mass was therefore established as a covalent adduct of the protein with 2-mercaptoethanol (+76 Da). Reduction and alkylation studies, followed by isolation of tryptic peptides, established that the site of attachment was Cys36. Although no significant effect of the modification on the activity of the protein was observed, the study underlines the ease with which a protein can be modified covalently by a simple and mild purification procedure; such labelling, which may not always be benign, would be undetectable without the routine use of mass spectrometric analysis.Keywords. Fructose-l,6-bisphosphate aldolase ; mass spectrometry ; 2-mercaptoethanol ; cysteine residue ; protein chemistry.Aldolases can be classified into two groups depending on their molecular properties and their catalytic mechanism [l, 21. The most widely studied are the class-I fmctose-I ,6-bisphosphate aldolases (FBP-aldolases). This enzyme catalyses the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. The catalytic mechanism of the class-I aldolases proceeds via a carbanion intermediate stabilised by a protonated Schiff base formed between the substrate and an active-site lysine residue [3-51. In contrast, the class-I1 enzymes utilise a bound metal ion to fulfil a similar role 161.X-ray structures of a number of class-I aldolases, including the FBP-aldolases from rabbit 17, 81 and human tissues [9, 101 and from Drosophilu [ I l l have been determined. They are tetramers of molecular mass around 160kDa and are members of the a/p barrel family of enzymes. Comparatively little is known about the class-I1 aldolases. They are dimers of molecular mass around 80kDa and bind one zinc atodsubunit. The only detailed structural information available for a class-I1 aldolase is for the fuculose-I-phosphate aldolase of Escherichia coli [12]. Unlike the class-I enzymes, this enzyme does not adopt the alp barrel structure.In order to increase our understanding of the class-I1 aldolases, we have developed an expression system for the class- I1 FBP-aldolase from E. coli [13] which yields large amounts (approximately 1 g/l in crude extracts) of the enzyme, and a program of site-directed mutagenesis 1131 and X-ray crystallographic analysis has ...

Proteome analysis and comparison of Clostridium acetobutylicum ATCC 824 and Spo0A strain variants

“…Fba class II has one gene in the genome that could encode this protein, and one protein spot was identified. The E. coli and B. subtilis Fba homologs are post-translationally modified [26,32] suggesting that C. acetobutylicum may have another Fba variant, as yet unidentified. RpoA is part of the core RNA polymerase that transcribes genes specified by r factor promoter recognition and thus is important for regulating gene expression [34].…”

Identification of arginine 331 as an important active site residue in the Class II fructose‐1,6‐bisphosphate aldolase of Escherichia coli

“…Samples containing enzyme were pooled, assayed, and the radioactivity of each sample was measured using a scintillation counter. The amount of protein recovered in each sample was determined by amino acid analyses performed in duplicate (Packman & Berry, 1995). , 1989), Campylobacter jejuni (Burucoa et al, 1995), Schizosaccharomycespombe (Mutoh & Hayashi, 1994), Saccharomyces cerevisiue (Schwelberger et al, 1989), Neurospora crassa (Yamashita & Stuart, 1995), Corynebacterium glutamicum (von der Osten et al, 1989), Bacillussubtilis (Trach et al, 1988;Mitchell et al, 1992).…”

“…Samples containing enzyme were pooled, assayed, and the radioactivity of each sample was measured using a scintillation counter. The amount of protein recovered in each sample was determined by amino acid analyses performed in duplicate (Packman & Berry, 1995).…”

“…No crystal structure is yet available for a Class I1 FBP-aldolase, and so it remains unclear whether, and how, the mode of substrate binding is different in the two classes. As part of a study of the use of protein engineering to produce novel enzyme activities with use in biotechnology, the Class I1 FBPaldolase of Escherichia coli has been cloned (Alefounder et al, 1989) and a program of X-ray crystallography and characterization has been initiated (Naismith et al, 1992;Berry & Marshall, 1993;Kitagawa et al, 1995;Packman & Berry, 1995). In this paper, we extend this characterization to a study of the substrate binding site.…”

Reactivity of the NS2/3(907–1206)ASK4protein with β‐mercaptoethanol studied by electrospray ion trap mass spectrometry

“…However, some modifications can occur during routine protein handling and can lead to a decrease in enzyme activity. Some examples are oxidation of methionine,2 deamidation of asparagine,3–5 cysteine modification with acrylamide for gel‐purified proteins6–8 and with β‐mercaptoethanol in solution 9–12…”