TABLE 2.
Kinetic parameters of mKAT III toward amino acids and α-keto acidsa
| Substrate | Km (mM) | kcat (min−1) | kcat/Km (min−1 mM−1) |
|---|---|---|---|
| Amino acid substrates | |||
| Glutamine | 0.7 ± 0.2 | 136.0 ± 14.0 | 194.2 |
| Histidine | 0.7 ± 0.4 | 120.0 ± 20.0 | 171.4 |
| Methionine | 0.9 ± 0.7 | 146.0 ± 50.0 | 162.2 |
| Phenylalanine | 1.1 ± 0.4 | 162.0 ± 20.0 | 147.2 |
| Asparagine | 1.4 ± 0.4 | 176.0 ± 18.0 | 125.7 |
| Cysteine | 0.7 ± 0.4 | 78.0 ± 12.0 | 111.4 |
| Kynurenine | 1.5 ± 0.5 | 138.0 ± 18.0 | 92.0 |
| Serine | 3.0 ± 0.7 | 130.0 ± 10.0 | 43.3 |
| Tryptophan | 7.1 ± 4.2 | 213.9 ± 60.0 | 30.1 |
| Tyrosine | 2.7 ± 0.9 | 62.0 ± 8.0 | 23.0 |
| Alanine | 6.2 ± 2.5 | 92.0 ± 21.9 | 14.8 |
| Keto acid substrates | |||
| Glyoxylate | 0.4 ± 0.2 | 162.8 ± 35.4 | 407.0 |
| α-Ketocaproic acid | 0.5 ± 0.2 | 155.3 ± 16.2 | 310.6 |
| Phenylpyruvate | 0.6 ± 0.3 | 152.0 ± 27.3 | 253.3 |
| α-Ketobutyrate | 1.0 ± 0.1 | 175.5 ± 7.4 | 175.5 |
| αKMB | 0.4 ± 0.2 | 60.9 ± 11.9 | 152.3 |
| α-Ketovalerate | 1.2 ± 0.3 | 162.2 ± 11.5 | 135.27 |
| Indo-3-pyruvate | 0.5 ± 0.5 | 62.0 ± 13.0 | 124.0 |
| Hydroxy-phenylpyruvate | 1.0 ± 0.1 | 88.9 ± 5.0 | 88.9 |
| Mercaptopyruvate | 2.4 ± 0.5 | 196.5 ± 13.6 | 81.9 |
| Oxaloacetate | 4.9 ± 1.2 | 220.7 ± 27.6 | 45.0 |
| Pyruvate | 10.6 ± 3.8 | 112.3 ± 16.9 | 10.6 |
| α-Ketoisocaproic acid | 5.3 ± 1.4 | 44.4 ± 6.3 | 8.4 |
| α-Ketoglutarate | 8.1 ± 12.8 | 22.2 ± 11.7 | 2.7 |
a
The activities were measured as described in Materials and Methods. The Km and kcat values for keto acids were derived by using various concentrations of individual keto acids in the presence of 10 mM kynurenine; for amino acids, the data were derived by using various concentrations of individual amino acids in the presence of 2 mM glyoxylate. The parameters were calculated by fitting the Michaelis-Menten equation to the experimental data, using an enzyme kinetics module.