TABLE 3.
Specific activities of enzymes of the BCAA pathway
| Strain | Mediuma | Sp act (nmol min−1 mg of protein−1) ofb:
|
|||||
|---|---|---|---|---|---|---|---|
| TDc | AHAS Id,e | AHAS IId | AHAS IIIe | KARI | DH | ||
| S. typhimurium | |||||||
| LT2 | Glc | 289 ± 40 | 8.9 ± 0.1 | 14.0 ± 0.3 | 7.8 ± 2.6 | 57 | |
| Glc + i.l.v. | 42 | 5.6 | 4.6 | 4.1 | NM | ||
| TV105 | Glc | 528 ± 20 | 0 | 30.4 ± 7.0 | 11.0 ± 0.7 | 97 | |
| Glc + i.l.v. | 35 | 0 | 3.7 | 4.5 | NM | ||
| TV506 | Glc | 659 ± 20 | 0 | 32.6 ± 2.2 | 8.3 ± 1.4 | 56 | |
| TV496 | Glc + Pan | 512 ± 68 | 31.3 ± 0.3 | 0 | 7.6 ± 1.2 | 101 | |
| Glc + Ile | 449 ± 70 | 31.3 ± 0.5 | 0 | 10.1 ± 1.2 | 115 | ||
| Glc + i.l.v. | 168 | 3.9 | 0 | 4.6 | NM | ||
| TV497 | Glc + Pan | 548 ± 98 | 34.7 ± 0.7 | 0 | 5.9 ± 1.7 | 156 | |
| TV108 | Glc + Ile | 21 | 61.0 | 0 | 9.3 | 1.4 | |
| TV493 | Glc + Ile, Val | 795 ± 46 | 0 | 0 | 4.5 ± 1.5 | NM | |
| TV503 | Glc + Ile, Val | 416 ± 23 | 0 | 0 | 4.7 ± 0.8 | NM | |
| LT2 | Acetate | 282 ± 14 | 27.0 ± 2.5 | 11.5 ± 0.9 | 9.6 ± 0.9 | 71 | |
| TV105 | Acetate | 510 ± 90 | 0 | 34.0 ± 5.0 | 13.3 ± 3.1 | 126 | |
| TV496 | Acetate | 334 ± 50 | 62.0 ± 6.0 | 0 | 6.4 ± 1.0 | 57 | |
| TV497 | Acetate + Pan | NM | 170.0 | 0 | 9.2 | NM | |
| TV108 | Acetate | 35 | 148.0 | 0 | 9.1 | NM | |
| E. coli K-12 MM294 | Glc | 104 ± 10 | 18.7 ± 3.0 | 15.4 ± 4.0 | 25.0 ± 3.2 | 25 | |
| Acetate | 145 ± 35 | 58.4 ± 6.6 | 37.7 ± 7.5 | 17.0 ± 4.0 | 8.4 | ||
The cells were grown in glucose (Glc) or acetate as described in Table 2, footnote a. Additions to the media were as follows: i.l.v., 0.38 mM each isoleucine, valine, and leucine (repressing conditions); Val, 0.85 mM valine; Ile, 0.38 mM isoleucine; and Pan, 0.34 mM pantothenate.
The enzyme activities were determined by standard methods as described in Materials and Methods. TD, threonine deaminase (EC 4.2.1.16); KARI, ketol acid reductoisomerase (EC 1.1.1.86); DH, dihydroxy acid dehydratase (EC 4.2.1.19). NM, not measured.
The determination of TD also included the addition of 0.1 to 1.4 mM Ile to assay mixtures to verify the presence of the normal ilvA gene product or the described ilvA219 mutation leading to feedback insensitivity.
AHAS isozymes I and II of S. typhimurium were determined differentially on the basis of their differential inhibition by Val and sulfometuron methyl as described in Materials and Methods. The levels of an isozyme were assumed to be zero in a strain deficient in the gene for that enzyme.
AHAS isozymes I and III of E. coli were determined differentially on the basis of their differential dependence on FAD (see Materials and Methods). Excess small subunits of AHAS III were also added to the assay mixtures (see Results).