Table 2. Microscopic rate constants for elementary steps in the reactions of WT AHAS II and three mutants.
| Enzyme | Reaction* | kcat, s-1 | k′2, s-1 | k′3, s-1 | k′4, s-1 | k′5, s-1 |
|---|---|---|---|---|---|---|
| WT | Pyr + Pyr | 20 ± 0.2 | 24 ± 4 | 530 ± 70 | 1,060 ± 175 | 176 ± 36 |
| Pyr + 2KB | 20 ± 0.2 | 21 ± 4 | 399 ± 54 | >2,000 | >2,000 | |
| Met250Ala | Pyr + Pyr | 3 ± 0.1 | 28 ± 7 | 35 ± 7 | 11.1 ± 1.3 | 5.6 ± 0.3 |
| Pyr + 2KB | 25 ± 0.2 | 42 ± 3 | 87 ± 16 | 262 ± 81 | >1,050 | |
| Arg276Lys | Pyr + Pyr | 4.6 ± 0.1 | 9.3 ± 1.4 | >75 | 15 ± 2 | 35 ± 3 |
| Pyr + 2KB | 9.1 ± 0.2 | 14.1 ± 1.5 | >288 | 57 ± 8 | 48 ± 7 | |
| Trp464Leu | Pyr + Pyr | 13 ± 0.1 | 16.4 ± 1.1 | 234 ± 68 | 172 ± 44 | 140 ± 37 |
| (Pyr + 2KB) ⇒ AHA† | 13 ± 0.1 | 16.2 ± 1.2 | 208 ± 49 | 310 ± 81 | 180 ± 47 | |
| (Pyr + 2KB) ⇒ AHB† | ≈360 | ≈210 | ||||
| (Pyr + Pyr) ⇒ AL† | ≈120 | ≈140 |
Net rate constants (see (Fig. 2) for formation of lactyl-ThDP from bound pyruvate (k′2), decarboxylation of lactyl-ThDP (k′3), covalent ligation of the second ketoacid (k′4), and release of AHA product (k′5) were calculated by using Eqs. 6, 7, 8, 9 from kcat and the distribution of ThDP-bound intermediates, determined by the rapid mixing-quench/NMR method at 37°C in 0.1 M KPi (pH 7.6). The constants for the WT enzyme are described in ref. 9.
Pyr + Pyr is the formation of AL from two molecules of pyruvate, in the presence of 100 mM pyruvate. Pyr + 2KB is the formation of AHB from pyruvate and 2-ketobutyrate, in the presence of 50 mM of each. Under these conditions, <2% of the reaction catalyzed by WT, Met250Ala, or Arg276Lys enzymes goes to AL
Pyr + 2KB ⇒ AHA is the formation of the total of acetohydroxyacids [Σ(AL + AHB)] in the presence of equimolar amounts of pyruvate and 2-ketobutyrate (50 mM each). The separate rates for formation of AHB and AL under these conditions are estimated on the following two rows of the table