Figure 1. In vitro labelling of purified E. faecalis proteins with [γ-³²P]ATP. Modification reactions were carried out in the presence of E. faecalis crude extracts and the different proteins (1 μg of YtxH-like protein, 3 μg of the other proteins) using two different compositions of divalent cations. The assay mixtures were separated by electrophoresis on SDS/polyacrylamide gels. A, Coomassie blue-stained gel. Lanes 1–5, experiments in the presence of 10 mM MgCl₂: lane 1, YtxH-like protein; lane 2, enolase; lane 3, GapA, lane 4 GapB; lane 5, control experiment without a purified protein. Lanes 6–10, experiments in the presence of 5 mM MgCl₂, 1 mM MnCl₂ and 1 mM ZnCl₂: lane 6, control experiment without a purified protein; lane 7, YtxH-like protein; lane 8, enolase; lane 9, GapA; lane 10 GapB. B, Autoradiogram of the Coomassie blue-stained gel.

Figure 2. In vivo labelling of His-tagged enolase in E. coli NM522[pGBenoEc] and a control strain carrying pQE30 without an insert grown in [³³P]phosphate-containing medium. Centrifuged cells from 0.5 ml cultures were dissolved in 30 μl of SDS sample buffer and 15 μl aliquots were separated by electrophoresis on SDS/polyacrylamide gels. A, Coomassie blue-stained gel: lane 1, control experiment with NM522[pQE30]; lane 2, same sample as lane 1 heated for ten minutes to 100 °C prior to electrophoresis; lane 3, crude extract of NM522[pGBenoEc]; lane 4, same sample as lane 3 heated to 100 °C; lane 5, M_r standards. B, Autoradiogram of the Coomassie blue-stained gel.

Figure 3. Effect of glycolytic intermediates on in vitro labelling of E. faecalis enolase. A, Autoradiogram of an SDS/polyacrylamide gel on which E. faecalis enolase labelled in vitro with [γ-³²P]ATP in the presence of E. faecalis crude extracts and various glycolytic intermediates (5 mM final concentration) had been separated by electrophoresis. Lane 1, without glycolytic intermediates; lane 2, pyruvate; lane 3, 2-PG; lane 4, PEP; and lane 5, 3-PG. B, Autoradiogram of SDS/polyacrylamide gels on which E. faecalis enolase labelled in vitro with 10 μM [γ-³²P]ATP or 15 μM [³²P]PEP had been separated. Modification in the presence of: lane 1, [γ-³²P]ATP and 5 mM MgCl₂; lane 2, [γ-³²P]ATP, 5 mM MgCl₂ and 5 mM pyruvate; lane 3, [³²P]PEP and 5 mM MgCl₂; lane 4, [³²P]PEP and 5 mM MnCl₂; lane 5, [³²P]PEP and 1 mM EDTA.

Figure 4. Time dependence of the modification reaction with enolases from bacteria, yeast and rabbit. Enolase (5 μg) from either E. faecalis, E. coli, B. subtilis, S. cerevisiae or rabbit muscle was incubated with 15 μM [³²P]PEP at 37 °C. Aliquots were withdrawn at the indicated time intervals and separated by electrophoresis on SDS/polyacrylamide gels, which were dried and exposed to autoradiography.

Figure 5. Labelling of enolase from E. faecalis, E. coli and S. cerevisiae with 1 or 2-[¹⁴C]PEP. Purified enolases were incubated for one hour at 37 °C with 1 or 2-[¹⁴C]PEP. The assay mixtures were separated by electrophoresis on SDS/polyacrylamide gels, which were dried and exposed to autoradiography. Experiments were carried out with: lane 1, 2-[¹⁴C]pyruvate; lane 2, 2-[¹⁴C]PEP; lane 3, 1-[¹⁴C]PEP; lane 4, 1-[¹⁴C]pyruvate.

Figure 6. Separation of modified and unmodified enolase on urea/polyacrylamide gels. A, Portions (5 μg) of (lane 1) in vivo ³³P-labelled Ni-NTA-purified and (lane 2) in vitro [³²P]PEP-modified E. coli enolase were separated by electrophoresis on a urea/polyacrylamide gel, which was stained with Coomassie blue. Lanes 3 and 4, autoradiogram of the Coomassie blue-stained gel. B, A portion (7 μg) of HPLC-purified unmodified enolase (lane 1) was incubated with various amounts of 2-PG (lanes 2–7) and PEP (lanes 8 and 9) before separating the samples on a urea/polyacrylamide gel, which was stained with Coomassie blue.

Figure 7. Enzyme activity of modified and unmodified E. coli enolase. A, Electrophoresis of the fractions obtained after separation of in vitro modified E. coli enolase by HPLC on a semipreparative C8 reverse-phase column on a urea/polyacrylamide gel. B, Enolase activity assays. To carry out enolase tests with similar amounts of protein, the following aliquots of the HPLC fractions were added to the assay mixtures: 20 μl of fraction 4; 6 μl of fraction 5; 2.6 μl of fraction 6; 2.8 μl of fraction 7 and 1.4 μl of fraction 8.

Figure 8. Mass spectrometry and collision-induced dissociation with the singly charged small chymotryptic modified peptide (m/z=888.5) derived from E. coli enolase. The peaks corresponding to the different b and y fragments and to the fragments that had lost phosphoric acid, 2-PG or water, are indicated. The b fragments extend from the N-terminal Ser337 to the break point, while the y fragments extend from the C-terminal Phe342 to the break point. Peaks marked with a * correspond to fragments containing modified Lys341. For example, fragmentation after the second isoleucine produced peptides b4 (S-I-L-I) and y2^* (K^*-F), fragmentation after leucine produced peptides b3 (S-I-L) and y3^* (I-K^*-F), etc.

Figure 9. Mutations preventing the export of enolase into the medium. E. coli wild-type and mutant enolases (E167Q, K341R and K341E) were synthesised in E. coli cells without induction by IPTG. A, Coomassie blue-stained SDS/polyacrylamide gel. Similar amounts of enolase were present in crude extracts prepared from NM522 cells carrying the coresponding plasmids: lane 1, M_r standard; lane 2, wild-type enolase; lane 3, E167Q; lane 4, K341R and lane 5, K341E mutant enolases. B, Coomassie blue-stained urea/polyacrylamide gel. Wild-type and several mutant enolases were purified from cellular crude extracts and their growth media by Ni-NTA chromatography. Wild-type enolase isolated from: lane 1, crude extract; lane 2, growth medium. E167Q mutant enolase from: lane 3, crude extract; lane 4, growth medium. K341R mutant enolase from: lane 5, crude extract; lane 6, growth medium. K341E mutant enolase from: lane 7, crude extract; lane 8, growth medium. Owing to the replacement of the positively charged Lys341 with a negatively charged glutamate, the K341E mutant enolase migrated slightly faster on urea gels than the wild-type enzyme. C, Coomassie blue-stained urea/polyacrylamide gel to test the demodification of wild-type enolase: HPLC-enriched modified enolase was incubated in the presence of 1 mM MgCl₂ for various time periods at 37 °C before the samples were separated on a urea/polyacrylamide gel, which was stained with Coomassie blue.

Table 1. Comparison of the specific activities of wild-type and mutant enolases measured at saturating (2 mM) 2-PG concentration

Table 2. PCR primers used in this study for amplification and mutagenesis