(+-)-Methionine → (+-)-Methionine

HRID 2265782

Reaction equation

HRID 2265782 reaction equation
[Na+].[Na+].[P:3]([O:15][CH2:16][C@H:17]1[O:21][C@@H:20]([N:22]2[C:31]3[N:30]=[CH:29][N:28]=[C:26]([NH2:27])[C:25]=3[N:24]=[CH:23]2)[C@H:19]([OH:32])[C@@H:18]1[OH:33])([O:6][P:7]([O:10][P:11]([OH:14])([OH:13])=[O:12])([O-:9])=[O:8])(=[O:5])[O-:4].[NH2:34][CH:35]([C:40]([OH:42])=[O:41])[CH2:36][CH2:37][S:38][CH3:39].C(O)C(N)(CO)CO.Cl.[Cl-].[K+]>ClC(Cl)(Cl)C(O)=O>[NH2:34][C@H:35]([C:40](=[O:41])[OH:42])[CH2:36][CH2:37][S+:38]([CH3:39])[CH2:16][C@H:17]1[O:21][C@@H:20]([N:22]2[C:31]3[N:30]=[CH:29][N:28]=[C:26]([NH2:27])[C:25]=3[N:24]=[CH:23]2)[C@H:19]([OH:32])[C@@H:18]1[OH:33].[P:3]([O:15][CH2:16][C@H:17]1[O:21][C@@H:20]([N:22]2[C:31]3[N:30]=[CH:29][N:28]=[C:26]([NH2:27])[C:25]=3[N:24]=[CH:23]2)[C@H:19]([OH:32])[C@@H:18]1[OH:33])([O:6][P:7]([O:10][P:11]([OH:13])([OH:14])=[O:12])([OH:9])=[O:8])(=[O:4])[OH:5].[NH2:34][CH:35]([C:40]([OH:42])=[O:41])[CH2:36][CH2:37][S:38][CH3:39]

Participants

8

Reactant 4

HCID 876(+-)-MethionineHCID 4873Potassium ChlorideHCID 93573Tromethamine hydrochlorideHCID 2723595ATP (Adenosine-Triphosphate)

Product 3

HCID 876(+-)-MethionineHCID 59575'-AtpHCID 34756S-adenosyl-L-methionine

Reagents, catalysts & solvents 1

HCID 6421Trichloroacetic AcidSolvent

Procedure

Prepare substrate solution A containing 8 mM adenosine triphosphate disodium (ATP), 32 mM DL-methionine, 100 mM Tris hydrochloride buffer, 100 mM KCl (final concentration) and 20 mM MgCl2 (final concentration) and adjusted pH to 7.5. Two hundred and fifty μl of the prepared substrate solution A was mixed with 150 μl sterile distilled water, 100 μl of the S-adenosylmethionine synthetase prepared according to Example 7, incubated at 58° C. for 20 min. The reaction was stopped by adding 300 μl 10% trichloroacetic acid (TCA), and then centrifuged at 17,800 g for 15 min at 10° C. to collect the supernatant as S-adenosylmethionine. The quantity of S-adenosylmethionine was determined by high pressure liquid chromatography (HPLC). HPLC column: C18 reverse column (Waters, USA); mobile phase: acetonitrile:water (85:15); flow rate: 0.5 mL/min; detector: ELSD 500. Injection volume was 10 μl. Protein concentration was determined using Coomassie® Plus Protein Assay Reagent Kit (Pierce, USA) and SDS-PAGE. One unit of enzyme specific activity is defined as the amount of enzyme that produces S-adenosylmethionine from 1 μmole of adenosine triphosphate (ATP) and 1 μmole of DL-methionine per min under the above described conditions. Table 2 below shows the specific activity of wild-type S-adenosylmethionine synthetase.

Workup

  1. concentrationconcentration) and 20 mM MgCl2 (final
  2. concentrationconcentration) and
  3. additionTwo hundred and fifty μl of the prepared substrate solution A was mixed with 150 μl
  4. distillationsterile distilled water, 100 μl of the S-adenosylmethionine synthetase
  5. customprepared
  6. waitcentrifuged at 17,800 g for 15 min at 10° C.
  7. customto collect the supernatant as S-adenosylmethionine
  8. concentrationProtein concentration

Sources & evidence

ORD record
ord-884e4fac234142c4894ceca2755c55e2
Source dataset
uspto-grants-2010_12
Patent number
US07858351B2

Linked notes

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