Chemical-modification rescue assessed by mass spectrometry demonstrates that gamma-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 solvent-accessible 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 gamma-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.

Fig. 1.

Chemical modification rescue of tetK107C. (A) Modification of tetK107C with BrEA monitored by recovery of specific activity in units aldolase activity/mg. Conditions: 50 mM BrEA, 200 mM AMPSO, pH 8.9, 0.01 mM protein, 25°C. (•), Unreacted tetK107C; (▴), tetK107C–EA; (♦), gtet–EA. (B) Fourier-transform spectrum of ESI-FTMS of unreacted tetK107C. (C) Deconvolution spectrum of (B). (D) Deconvolution spectrum of aminoethylated tetK107C–EA. Modification conditions: 50 mM BrEA, 200 mM Tris-Cl, pH 8.9, 20°C, 20 h. Protein samples introduced into the mass spectrometer were 20 μM aldolase in 100 mM NH₄OAc/1% formic acid.

Fig. 2.

Mass spectrum of modified, digested tetK107C–EA. ESI-FTMS of tetK107C–EA trypsin peptides double-labeled with 1:1 d₀/d₄-BrEA (100 mM total) as in Figure 1D ▶. For clarity, only major peaks are labeled with assignments defined by the method of Chowdhury et al. (1990). (Inset) Overlapping peak sets of T₂₆ and T₂₉ and methionine oxidation peak, T₂₆-Met-ox (see Table 3). The effective concentration of d₄-BrEA was determined by comparing the rate of activity rescue to that of commercial BrEA (d₀-BrEA) determined at known concentrations.

Fig. 3.

Tryptic peptide map of tetK107C-EA aldolase. Assigned tryptic fragments are depicted by shaded boxes. Light-shaded boxes are completely digested peptides and dark-shaded boxes are incompletely digested peptides. Single arrows denote cysteine positions, and double arrows denote histidine or methionine positions. Amino acid is derived from the DNA sequence of the gtet expression plasmid with numbering as defined by Tolan et al. (1984). Nomenclature for tryptic peptides as defined in Figure 2 ▶ (Chowdhury et al. 1990).

Fig. 4.

Mass spectra of selected ions from tryptic digest of tetK107C–EA. (A) Double labeling of C107-containing T₁₄ peptide under native conditions. The d₀ bracket denotes isotope peaks of T₁₄ reacted with Br-CH₂-CH₂-NH₃⁺ (50 mM) and d₄ bracket denotes reaction with Br-CD₂-CD₂-NH₃⁺ (50 mM). (B) Material from (A) was denatured and reacted with d₀-BrEA (100 mM). (C) Two regions of m/z spectrum corresponding to positions of unmodified (m/z 462.29) and modified (m/z 505.33) T₂₀ peptide under native conditions. (D) Sample treated as for (C) after subsequent labeling under denaturing conditions. (E) Peptides, T₁ and T₂₁, exhibiting aminoethylation at residues other than cysteine under native conditions. (F) As in (E), except under denaturing conditions.