Table 2.
Kinetic parameters of selected KARI homologues against non-native substrates
| Substrate | Kinetic parametersa | Sco1 | Sco2 | Sli1 | Sli2 | Sam | Enzymec Sav | Sgr | Spr | Svi2 | Cgl | P5CR |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| MAA | kcat (s−1) | 1 ± 0.2 | 0.6 ± 0.05 | 0.675 ± 0.06 | 0.5 ± 0.05 | 1.2 ± 0.09 | 1.8 ± 0.1 | 1.05 ± 0.1 | 2 ± 0.2 | 2 ± 0.2 | 0.4 ± 0.03 | NDb |
| KM (mM) | 50 ± 4 | 60 ± 5 | 45 ± 4 | 50 ± 4 | 40 ± 4 | 30 ± 2 | 35 ± 4 | 40 ± 5 | 50 ± 5 | 40 ± 1.5 | NDb | |
| kcat/ KM(M−1 s−1) | 20 ± 1.8 | 10 ± 1 | 15 ± 1.2 | 10 ± 0.9 | 30 ± 3 | 60 ± 7 | 30 ± 2.5 | 50 ± 6 | 40 ± 5 | 10 ± 1.3 | NDb | |
| HP | kcat (s−1) | NDb | 0.5 ± 0.05 | NDb | 0.7 ± 0.06 | 1 ± 0.09 | 2 ± 0.2 | 1.5 ± 0.1 | 2 ± 0.2 | 2 ± 0.2 | 0.5 ± 0.04 | NDb |
| KM (mM) | NDb | 40 ± 3 | NDb | 50 ± 5 | 50 ± 4 | 60 ± 5 | 30 ± 4 | 60 ± 5 | 50 ± 5 | 40 ± 4 | NDb | |
| kcat/ KM(M−1 s−1) | NDb | 60 ± 7 | NDb | 40 ± 3.5 | 20 ± 2.2 | 50 ± 5 | 40 ± 3.8 | 30 ± 3 | 50 ± 4 | 60 ± 3.9 | NDb | |
| Pyr | kcat (s−1) | 2.8 ± 0.05 | 5.6 ± 0.5 | 6.7 ± 0.7 | 3 ± 0.2 | 6.3 ± 0.5 | 3.6 ± 0.4 | 1 ± 0.1 | 3 ± 0.2 | 5.7 ± 0.5 | 8.6 ± 0.9 | NDb |
| KM (mM) | 950 ± 80 | 700 ± 70 | 1120 ± 110 | 1010 ± 100 | 1050 ± 99 | 1200 ± 110 | 1060 ± 120 | 1080 ± 100 | 1140 ± 112 | 1070 ± 118 | NDb | |
| kcat/ KM(M−1 s−1) | 3 ± 0.2 | 8 ± 0.7 | 6 ± 0.5 | 3 ± 0.2 | 6 ± 0.5 | 3 ± 0.3 | 1 ± 0.2 | 3 ± 0.3 | 5 ± 0.4 | 8 ± 0.7 | NDb |
Kinetic parameters shown are means and standard errors of three enzymatic reaction.
ND, activity not determined because it is below the limit of detection of the enzyme assay, which is kcat/KM = 0.000013 M−1s−1.
Enzymes nomenclature: Eco: KARI, ilvC, Escherichia coli; Sco1: KARI1, ilvC1, Streptomyces coelicolor; Sco2: KARI2, ilvC2, Streptomyces coelicolor: Sli1: KARI1, ilvC1, Streptomyces lividans; Sli2: KARI2, ilvC2, Streptomyces lividans; Sam: KARI, ilvC, Streptomyces ambofaciens; Sav: KARI, ilvC, Streptomyces avermitilis; Sgr: KARI, ilvC, Streptomyces griseus; Spr: KARI, ilvC, Streptomyces pristinaespiralis; Svi2: KARI2, ilvC2, Streptomyces viridifaciens; Cgl: KARI, ilvC-panE, Corynebacterium glutamicum; P5CR: P5CR, proC, Streptomyces coelicolor.