Thiamine Diphosphate → 2-Acetoxypropionic Acid

HRID 1591032Notes

Reaction equation

HRID 1591032 reaction equation
N([C:6]([CH2:11][OH:12])([CH2:9]O)CO)[CH2:2][C:3]([OH:5])=[O:4].[OH-:13].[Na+].[Mg+2].[Cl-].[Cl-].CC1[N+](CC2C(N)=NC(C)=NC=2)=CS[C:20]=1[CH2:33][CH2:34][O:35]P(OP(O)(O)=O)(O)=O.[Cl-]>>[CH3:20][CH:33]([O:5][C:3]([CH3:2])=[O:4])[C:34]([OH:35])=[O:13].[O:12]=[C:11]([CH2:6][CH3:9])[C:34]([O-:35])=[O:13]

Participants

5

Reactant 3

HCID 9068Thiamine DiphosphateHCID 5360315Magnesium ChlorideHCID 131717881HCID 131717881, compound

Product 2

HCID 790412-Acetoxypropionic AcidHCID 35932772-Ketobutyrate

Procedure

To a 1 ml solution containing 0.1 M tricine-NaOH, pH 8, 10 mM MgCl2, 0.1 mM thiamine pyrophosphate, and 0.1 mM flavin adenine dinucleotide were added various concentrations of pyruvate (10 μM to 10 mM), α-keto-butyrate (10 μM to 80 mM), or acetolactate (0.15 to 5.5 mM). After allowing the solution to equilibrate with air (21 % O2), the level of dissolved oxygen was monitored by use of a commercially available oxygen-sensitive electrode (Hansatech) and polarographic electronics as described by Sweet et al. (Analytical Biochemistry 107:337 (1980)). In the absence of further additions, negligible oxygen was consumed over a 30 minute period. To initiate the consumption of oxygen, 20 μl of a 2 mg/ml stock solution of acetolactate synthase was added if pyruvate or α-acetolactate were used as substrate, or 5 μl of a 49 mg/ml stock solution of acetolactate synthase was added if α-ketobutyrate was used as substrate. The limiting rate of oxygen consumption at saturating concentrations of pyruvate, α-ketobutyrate, or α-acetolactate are illustrated in Table 1. The concentrations of α-acetolactate or α-ketobutyrate that gave half of the maximum rate (the Michaelis constant) were 1.4 mM and 32 μM, respectively. The rate of oxygen consumption was constant over the range of concentrations of pyruvate examined, indicating that the Michaelis constant for this substrate was substantially lower than 10 μM.

Workup

  1. additionwere added various
  2. concentrationconcentrations of pyruvate (10 μM to 10 mM), α-keto-butyrate (10 μM to 80 mM), or acetolactate (0.15 to 5.5 mM)
  3. customIn the absence of further additions, negligible oxygen was consumed over a 30 minute period
  4. customthe consumption of oxygen, 20 μl of a 2 mg/ml stock solution of acetolactate synthase
  5. additionwas added if pyruvate or α-acetolactate
  6. additionwas added if α-ketobutyrate
  7. customThe limiting rate of oxygen consumption
  8. concentrationat saturating concentrations of pyruvate, α-ketobutyrate, or α-acetolactate

Sources & evidence

ORD record
ord-80193a3b071b4c639cad07099cb42b65
Source dataset
uspto-grants-1993_04
Patent number
US05206135

Reaction notes

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