Table 1.
Activities and product ratios of BcUGAepi variants reacted with UDP‐GlcA (1) and UDP‐GalA (2). The activities were determined from the linear part of the time course, excluding the initial burst present in some reactions (Figures S14–S22 and S28–S30). The slope of the linear regression (mM min−1) was divided by the enzyme concentration (mg mL−1) to give the initial rate in μmol(min mg)−1, which equates to U mg−1.
|
Enzyme |
Substrate |
Activity [mU mg−1] |
UDP‐GlcA (1, %) |
UDP‐GalA (2, %) |
UDP‐xylose (3, %) |
UDP‐4‐ketopentose (4, %) |
Burst[e] |
|---|---|---|---|---|---|---|---|
|
Wild type |
1 |
500 |
33 |
67 |
0 |
0 |
– |
|
T126A |
1 |
0.08 |
33[a] |
67[a] |
0[a] |
0[a] |
– |
|
S127A |
1 |
24.3 |
33 |
67 |
0 |
0 |
– |
|
S128A |
1 |
11.8 |
33 |
67 |
0 |
0 |
– |
|
S128E |
1 |
0.2 |
85.4[b] |
12.1[b] |
1.7[b] |
0.8[b] |
– |
|
T178A |
1 |
0.09 |
33[a] |
67[a] |
0[a] |
0[a] |
– |
|
R185A |
1 |
0.05 |
90.7[c] |
0[c] |
0[c] |
9.3[c] |
0.10 |
|
R185D |
1 |
0.05 (≈2.6) |
92.5[c] |
0[c] |
5.1[c] (≈95) |
2.4[c] (≈5) |
0.22 |
|
R185H |
1 |
0.05 (≈5) |
81.3c] |
8.1[c] |
9.0[c] (≈95) |
1.6[c] (≈5) |
2.43 |
|
R185K |
1 |
0.3 (≈66) |
51.1[c] |
21.8[c] |
26.8[c] (≈98) |
0.3[c] (≈2) |
– |
|
Wild type |
2 |
500 |
33 |
67 |
0 |
0 |
– |
|
T126A |
2 |
0.09 |
24.8 |
74.8 |
0 |
0.4 |
– |
|
S127A |
2 |
218 |
29.1 |
70.5 |
0.4[d] |
0 |
– |
|
R185H |
2 |
0.1 |
18.3 |
70.7 |
5.0[d] |
6.0 |
– |
Unless stated otherwise, the ratios are reported after 24 h of reaction time. For R185H, R185D, and R185K enzymes, the activity and composition of the initial product burst (at 1 min) are given in brackets. Each reaction was performed at least in duplicate and the data accuracy is ±5 %. [a] Product ratios are after 48 h reaction. [b] Product ratios are after 2 h reaction. [c] Product ratios are after 2 h 30 min reaction. [d] Product is UDP‐pentose (UDP‐xylose and/or UDP‐l‐arabinose). [e] Total molarity of product burst after 1 min (μM) divided by the enzyme molarity (μM). Burst refers here to a specific time of sampling to capture a fast release of an initial product. This sampling may not have allowed analysis of the first enzyme turnover, however (see the entry for the R185H variant). Note: specific activities were determined here in lieu of a full set of kinetic parameters (k cat, K m) given the focus on the analysis of the product formation.