Chemical‐modification rescue assessed by mass spectrometry demonstrates that γ‐thia‐lysine yields the same activity as lysine in aldolase

Abstract

The role of active site residues in fructose 1,6-bisphosphate aldolase is investigated by chemical-modification rescue. An active-site mutation, K107C, is constructed in a background where the four solventaccessible cysteine residues are converted to alanine. The resulting mutant, tetK107C, when reacted with bromoethylamine (BrEA), shows a 40-fold increase in activity (to 80% that of wild type). Determination of the sites and their degree of modification using electrospray ionization Fourier transform mass spectrometry (ESI-FTMS) is developed, allowing correlation of activity after chemical modification rescue to the degree of modification. The stoichiometry of the reaction is 2.5 aminoethylations per subunit, as measured by ESI-FTMS. Protein modification with a double-labeled mix (1:1) of natural abundance isotope (d 0 -BrEA) and 2-bromoethyl-1,1,2,2-d4-amine hydrobromide (d 4 -BrEA), followed by dialysis and trypsin digestion, shows aminoethylated peptides as "twin peptides" separated by four mass units in ESI-FTMS analysis. Using this detection procedure under nondenaturing (native) conditions, C107 is aminoethylated, whereas the four buried thiols remain unlabeled. Aminoethylation of other residues is observed, and correlates with those peptides containing histidine, methionine, and/or the amino terminus. Quantification of the aminoethylation reaction is achieved by labeling with nondeuterated d 0 -BrEA under denaturing conditions following double labeling under native conditions. In addition to complete labeling all five thiols, the intensity of the d 0 -BrEA peak for C107 containing peptides increases, and the change in the d 0 /d 4 ratio between native and denaturing conditions shows 82 ± 4.5% aminoethylation at C107. This correlation of modification with the recovered activity, indicates that ␥-thia-lysine replaces lysine in the catalytic mechanism. Kinetic constants measured for the rescued K107C mutant enzyme with the substrates fructose 1-phosphate and fructose 1,6-bisphosphate are consistent with the role of the positively charged lysine binding to the C6-phosphate. ESI-FTMS, combined with this double-labeling procedure, allows precise identification of sites and measurement of degree of protein modification.

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

“…Most importantly, this two-step procedure has established linkage junctions between various amino acids including the branched hydrophobic residues Leu and Val. [120] Although these transformations deliver non-native protein analogues of natural occurring amino acids, several studies have demonstrated they have similar biological function as their natural analogues. This auxiliary delivered the corresponding peptides in an analogous two-step protocol in very good yields even at very hindered Thr-Val junctions.…”

“…[117] Additionally, Gln or Glu derivatives 74 can be obtained by reaction with aiodoacetamide (75) [118] or a-iodoacetic acids (76, see also Section 7.2), [119] respectively, whereas Lys analogues 77 can be obtained by reaction with aziridines or bromoethylamines (78) (Scheme 12). [120] Although these transformations deliver non-native protein analogues of natural occurring amino acids, several studies have demonstrated they have similar biological function as their natural analogues. [119] In a very recent study the methylation of a Cys residue with methyl-4-nitrobenzenesulfonate was combined with an activation and intramolecular rearrangement with CNBr in formic acid followed by an O!N-acyl shift under slightly basic conditions (pH 7-8).…”

Clostridiolysin S, a Post-translationally Modified Biotoxin from Clostridium botulinum

“…This too failed to recover any of these peptides. Because ClosA and SagA have an array of contiguous cysteines within the primary sequence (CCCCSCCCC), another approach was taken based on some classic enzymological studies carried out on acetoacetate decarboxylase (30). This particular enzyme decarboxylates acetoacetate via the formation of an imine with an active site lysine.…”

From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

“…Notably, however, careful control of reaction conditions may prove necessary to prevent unwanted over‐reaction on non‐Cys residues (e.g., His. Met27). …”