Table 1.
Protein | kcat, min−1 | KmPRA, μM | kcat/KmPRA, M−1 s−1 | (A)KdrCdRP (B)KiProFAR, μM |
---|---|---|---|---|
Selected HisAF variants | ||||
HisAFselI | 0.57 | 84‡ | 113 | — |
HisAFselII | 0.017 | 53 | 5.4 | — |
HisAFselIII | 0.019 | 205‡ | 1.5 | — |
HisAFselIV | 0.081 | 67 | 20.2 | — |
Combined HisAF variants | ||||
HisAFcomI | 1.9 ± 0.1 | 19 ± 7 | (1.9 ± 0.7) × 103 | — |
HisAFcomII | 3 ± 1 | 14 ± 3§ | (3 ± 1) × 103 | 8.8B |
HisAFcomIII | 4.0 ± 0.4 | 2.9 ± 0.5 | (2.4 ± 0.5) × 104 | 5.6A/3.1B |
HisA variants | ||||
HisA-I* | 7 ± 2 | 35 ± 4 | (3.3 ± 0.7) × 103 | — |
HisA-II | 67 ± 4 | 16 ± 2 | (7.2 ± 0.8) × 104 | 4.5A/0.10B |
HisA-II-D8N | 0.020 ± 0.004 | 57 ± 24 | 8 ± 4 | — |
Wild-type enzymes | ||||
HisAF, HisA | <0.005 | — | — | — |
ecTrpF† | 2070 | 12 | 2.9 × 106 | 6.8A |
tmTrpF† | 222 | 0.28 | 1.3 × 107 | — |
Reaction conditions for all experiments were 50 mM Hepes, pH 7.5/4 mM MgCl2/4 mM EDTA/2 mM DTT/25 °C. All selected and combined HisAF variants contain the Y140H exchange to improve solubility. The variants HisAFselI, II, and IV additionally contain the V231M exchange (24). The shown values for the combined HisAF and HisA variants are the mean and SD, as deduced from at least 3 PRA saturation curves of different protein preparations. The values for the selected HisAF variants were deduced from single PRA saturation curves. The estimated error is less than factor 2.
*The HisA-I variant containing the exchanges H75Y, F111S, and D127V originally could not be saturated with PRA (13), probably due to the presence of residual orthophosphate, which is a competitive inhibitor of substrate binding. After extensive dialysis against phosphate-free buffer, we were able to record complete saturation curves and determine values for the Michaelis constant and the turnover number of HisA-I.
‡KmPRA values were determined in presence of >0.8 mM orthophosphate.
§KmPRA value was determined in presence of >0.1 mM orthophosphate.