Table 2.

Experimental design to test the activities of GLYAT and GLYATL1 with varying substrate concentrations.

Purpose	Constant Substrate	Variable Substrate
Evaluate the baseline enzyme activity of GLYAT and GLYATL1	Preferred amino acid	Preferred acyl-CoA
Evaluate the ability of GLYAT and GLYATL1 to conjugate isovaleryl-CoA as a substrate	Preferred amino acid	Isovaleryl-CoA
Determine the correlation between amino acid concentration and GLYAT and GLYATL1 reaction rate	Preferred acyl-CoA	Preferred amino acid
Determine the correlation between amino acid concentration and GLYAT and GLYATL1 reaction rate	Isovaleryl-CoA	Preferred amino acid

Amino acid substrates, when constant, were 20 mM whereas the preferred acyl-CoA substrates (benzoyl-CoA or phenylacetyl-CoA) were 250 µM and isovaleryl-CoA was 500 µM. Concentration variables for the acyl-CoA substrates were: 50, 100, 250, and 500 µM. Amino acid substrate concentrations were varied as follows: 0.5, 1, 2, 5, 10, and 20 mM.