Assay Method Information |
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| PEPCK Assay (PEP to OAA-allosteric) |
Description: | Allosteric binding site. When the enzyme was assayed in the direction of PEP →OAA, the standard reaction mixture contained 50 mM HEPES (pH 7.5), 2mMMnCl2, 10mMDTT, 0.5mMGDP, 2mMPEP, 187.5 μM NADH, 50 mM KHCO3, and 11 units of MDH. The reaction proceeded at 25 °C and was started with the addition of PEPCK (∼1−2 μg). Mutants were assayed as previously described with the exception that ~30 μg of enzyme was used to initiate the assay.14 Inhibition assays with MPA were conducted with varying concentrations of PEP with a fixed GDP concentration of 500 or 25 μM. Inhibition assays against GDP were conducted at a fixed concentration of PEP of 2 or 5 mM. Assays that did not contain MPA were conducted with methanol present at the identical concentration as a blank. |
Affinity data for this assay | |
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