Glyoxylate carboligase: a unique thiamin diphosphate-dependent enzyme that can cycle between the 4'-aminopyrimidinium and 1',4'-iminopyrimidine tautomeric forms in the absence of the conserved glutamate

Abstract

Glyoxylate carboligase (GCL) is a thiamin diphosphate (ThDP)-dependent enzyme, which catalyzes the decarboxylation of glyoxylate and ligation to a second molecule of glyoxylate to form tartronate semialdehyde (TSA). This enzyme is unique among ThDP enzymes in that it lacks a conserved glutamate near the N1' atom of ThDP (replaced by Val51) or any other potential acid-base side chains near ThDP. The V51D substitution shifts the pH optimum to 6.0-6.2 (pK(a) of 6.2) for TSA formation from pH 7.0-7.7 in wild-type GCL. This pK(a) is similar to the pK(a) of 6.1 for the 1',4'-iminopyrimidine (IP)-4'-aminopyrimidinium (APH(+)) protonic equilibrium, suggesting that the same groups control both ThDP protonation and TSA formation. The key covalent ThDP-bound intermediates were identified on V51D GCL by a combination of steady-state and stopped-flow circular dichroism methods, yielding rate constants for their formation and decomposition. It was demonstrated that active center variants with substitution at I393 could synthesize (S)-acetolactate from pyruvate solely, and acetylglycolate derived from pyruvate as the acetyl donor and glyoxylate as the acceptor, implying that this substitutent favored pyruvate as the donor in carboligase reactions. Consistent with these observations, the I393A GLC variants could stabilize the predecarboxylation intermediate analogues derived from acetylphosphinate, propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric forms notwithstanding the absence of the conserved glutamate. The role of the residue at the position occupied typically by the conserved Glu controls the pH dependence of kinetic parameters, while the entire reaction sequence could be catalyzed by ThDP itself, once the APH(+) form is accessible.

Figure 1

Structure of the active center of GCL demonstrating the hydrophobic environment of the thiazolium ring of ThDP. Figure was created using PyMOL based on the published structure of GCL (RCSB structure 2PAN) (5).

Figure 2

The effect of pH on CD spectra of V51D GCL. (Top) Near-UV CD spectra of V51D GCL at different pH values. The V51D GCL from stock was diluted to a concentration of 2.6 mg/mL (concentration of active centers = 40 μM) in 0.1 M KH₂PO₄ containing 0.5 mM ThDP and 2.5 mM MgCl₂. CD spectra were recorded after pH was adjusted to the desired value (pH 5.72 – pH 7.95). (Bottom) Dependence of the log CD₃₀₄ versus pH. Data were fitted to a single ionizing group (eq 1).

Figure 3

The effect of pH on TSA activity of GCL. (A) Progress curves of TSA formation were recorded at different pH values using CD at 290 nm. Conditions of the experiment are presented under Experimental Procedures. The linear part of the progress curves was used to calculate the initial velocity (CD₂₉₀/s) using the Pro-Data Viewer program. (B) Dependence of log (CD₂₉₀/s) versus pH. The logCD₂₉₀/s was plotted versus pH and data were fitted to eq (3) for two ionizing groups.

Figure 4

The effect of pH on TSA activity of V51D GCL. (Top) Progress curves of TSA formation at different pH values using CD at 290 nm. Conditions of the experiment are presented under Experimental Procedure. The linear part of the progress curves was used to calculate the initial velocity (CD₂₉₀/s) using the Pro-Data Viewer program. (Bottom) pH dependence of log (CD₂₉₀/s). Data were fitted to eq. (2) for a single ionizing group.

Figure 5

CD spectra of V51D GCL titrated by glyoxylate. The V51D GCL (1.23 mg/mL, concentration of active centers = 19.0 μM) in 0.1 M KH₂PO₄ (pH 6.5) containing 0.5 mM ThDP, 2.5 mM MgCl₂,1.0 mM DTT, 10 μM FAD and 1% glycerol was titrated by glyoxylate (0.010-8 mM) at 5 °C. CD spectra were recorded in the near-UV region after each addition of glyoxylate. Observable changes in the intensity of the CD band at 302 nm were detected with 1 mM, 5 mM and 8 mM glyoxylate, but not with 0.01-0.5 mM glyoxylate. On increasing the temperature to 20 °C, formation of (R)-TSA was evident.

Figure 6

ime course of the reaction of V51D GCL with glyoxylate monitored by stopped-flow CD. (A) Formation of 1′,4′-iminoglycolylThDP at pH 7.6 and 6 °C. V51D GCL (3.6 mg/mL, concentration of active centers = 55.6 μM) in 0.1 M KH₂PO₄ (pH 7.6) containing 0.5 mM ThDP, 2.5 mM MgCl₂, 1.0 mM DTT and 10 μM FAD in one syringe was mixed with an equal volume of 2.0 mM glyoxylate in the same buffer in the second syringe. Data were fitted to a double exponential equation (eq 4). (B) The decarboxylation of 1′,4′-iminoglycolyl-ThDP and the formation of TSA-ThDP complex at pH 7.6 and 15 °C. V51D GCL (2.3 mg/mL, concentration of active centers = 35.5 μM) in one syringe was mixed with 2.0 mM glyoxylate in the second syringe at 15 °C. (C) Time-dependent decarboxylation of 1′,4′-iminoglycolylThDP with data from Figure 5 (B) expanded. Data were fitted to a single exponential (eq 5). (D) The formation of TSA-ThDP complex and TSA release by V51D GCL at pH 7.6. The V51D GCL (1.7 mg/mL, concentration of active centers = 26 μM) was pre-incubated with 0.5 mM glyoxylate for 15 min in one syringe, and was then mixed at 6 °C with 16 mM glyoxylate in the second syringe. The data were fitted to a triple exponential (eq 6).

Figure 7

Reaction of V51D GCL with glyoxylate monitored by stopped-flow CD at pH 6.5. The V51D GCL (6.45 mg/mL, concentration of active centers = 99.6 μM) in 0.10 M KH₂PO₄ (pH 6.5) containing 0.5 mM ThDP, 2.5 mM MgCl₂, 1.0 mM DTT and 10 μM FAD in one syringe was mixed with an equal volume of 2 mM glyoxylate in the second syringe. The reaction was monitored over 45 s at 6 °C. Data were fitted to a triple exponential (eq 7)

Figure 8

CD titration of I393A GCL by pyruvate at 6 °C. (A) CD spectra of I393A GCL (2.0 mg/mL, concentration of active centers = 30.8 μM) in 0.1 M KH₂PO₄ (pH 7.6) containing 0.5 mM ThDP, 2.5 mM MgCl₂, 1.0 mM DTT and 10 μM FAD in the absence and in the presence of 0.020-0.70 mM pyruvate. Each spectrum was recorded after pre-incubation of I393A GCL with pyruvate for 20 min at 6 °C. (B) Dependence of the ellipticity at 302 nm on pyruvate concentration. Inset shows the plot of the CD₃₀₂ data points versus [pyruvate] at concentrations less than 0.80 mM. Data were fitted to a Hill equation (3).

Figure 9

Formation of an acetylglycolate product by I393A GCL as detected by CD. (Top) CD spectra of I393A GCL recorded in the presence of 1 mM pyruvate and on addition of 1 mM glyoxylate at 6 °C. The I393A GCL was diluted to a concentration of active centers of 30.8 μM in 0.1 M KH₂PO₄ (pH 7.6) containing 0.5 mM ThDP, 2.5 mM MgCl₂, 1.0 mM DTT, 10 μM FAD and 1% glycerol. (Middle) Time-dependent formation of an acetylglycolate-ThDP complex and product release. The I393A GCL (4.69 mg/mL, concentration of active centers = 72.4 μM) in buffer as in (Top) was pre-incubated with 1 mM pyruvate in one syringe and was then mixed at 6 °C with 1.0 mM glyoxylate in the second syringe. Data were fitted to a triple exponential (eq 7). (Bottom) Time-dependent release of a acetylglycolate product. Conditions were the same as in (Middle) but 4 mM glyoxylate was present in the second syringe.

Figure 10

CD spectra of I393A GCL titrated with AcPhi. (A)The I393A GCL (2.0 mg/mL, concentration of active centers = 30.8 μM) in 0.10 M KH₂PO₄ (pH 7.6) containing 5 mM MgCl₂ and 0.50 mM ThDP, was titrated by AcPhi (0.050-4.0 mM) at 25 °C. (B) Dependence of the CD at 302 nm on concentration of AcPhi. Data were fitted to a Hill equation (eq 3).

Scheme 1

A. Mechanism of GCL reaction, including tautomerization/ionization states of thiamin. The possible reactions of a decarboxylase-carboligase with glyoxylate. B. The possible reactions of a decarboxylase-carboligase with pyruvate as sole substrates, acetolactate is produced. With the I393A GCL, in the presence of pyruvate and glyoxylate the mixed product acetylglycolic acid is the expected product.

Scheme 2

Formation of covalent adducts of ThDP with pyruvate and with the pyruvate analogs MAP and AcPhi.

Scheme 3

Minimal mechanism and microscopic rate constants for V51D GCL