TABLE 2.
Kinetic characterization of wild-type and mutated forms of hUXS1
| Enzyme | kcat/limiting reactiona | Km UDP-GlcUA | Reaction productb | NADH formationc | Deuterium incorporation in product at C5d |
|---|---|---|---|---|---|
| s−1 | mm | ||||
| Wild-type | 0.2/oxidatione | 5.1 | 4, traces of 3 | ≤0.001 | Axialf |
| E120A | 1.7 × 10−3/reductione | 0.7 | 4, traces of 3g | ∼0.05g | Axialf |
| (3 and 4) | (∼0.5) | ||||
| Y147F | 1.3 × 10−4/NA | >30h | 3 | ∼1 | Axiali |
| R277Q | 1.5 × 10−4/NA | 0.1 | 3, traces of 4 | ∼1 | Double deuteration |
a Determined from the rate of conversion of UDP-GlcUA.
b Notation: 3, UDP-4-keto-pentose; 4, UDP-xylose.
c Molar ratio of NADH formed and UDP-GlcUA converted; data have S.D. of about 15%.
d Determined by 1H NMR.
e Determined by comparing the rate of NADH formation under conditions of rapid mixing in a stopped-flow apparatus to the steady-state rate of formation of UDP-xylose. Note that reduction is meant here to involve all reaction steps after formation of UDP-4-keto-GlcUA (2), including the decarboxylation. NA, not applicable.
f Stereochemical assignment is made for 4. It follows from the proposed enzymatic reaction that 3 will have deuterium incorporated at C5 with the same stereochemistry as 4.
g The molar ratio of 4 and 3 decreased in response to increase of UDP-GlcUA (1) concentration, from the shown value at 10 mm of 1 to the value in parentheses at 50 mm of 1.
h No exact determination possible, as no substrate saturation could be reached.
i Inferred from equivalence of 1H spectra of 3 formed by Y147F mutant and wild-type enzyme.