Quantitative Functional Characterization of Conserved Molecular Interactions in the Active Site of Mannitol 2‐Dehydrogenase
James E. Lucas ‡, Justin B. Siegel ‡,α,β
‡ Genome Center, University of California, Davis, CA 95616, USA
α Department of Chemistry, University of California, Davis, CA 95616, USA
β Department of Biochemistry & Molecular Medicine, University of California, Davis, CA 95616, USA
Received 5 January 2015; Accepted 2 March 2015
DOI 10.1002/pro.2669
First published online 2 April 2015
Lucas, J. E. and Siegel, J. B. (2015), Quantitative functional characterization of conserved molecular interactions in the active site of mannitol 2‐dehydrogenase. Protein Science, 24: 936–945. doi:10.1002/pro.2669
The three genes encoding active site mutants E133A, E133Q, and N300A contained unexpected mutations. We have reconstructed these mutants, sequence confirmed, and assayed the correct enzyme mutants on the reported substrates. Tables and figures with kinetic data for the corrected mutants are provided. We regret this error, and would like to thank AddGene for the due diligence carried out in order to identify this mistake.
Table 1.
Corrected Kinetic Constants for Mutant pfMDH on D‐Mannitol‡
| Enzyme | k cat (s−1) | KM (mM) | k cat/KM (M−1s−1) |
|---|---|---|---|
| E133A | 0.030 ± 0.001 | 1.99 ± 0.34 | 14.95 ± 2.60 |
| E133Q | 0.065 ± 0.001 | 3.96 ± 0.30 | 16.50 ± 1.27 |
| N300A | 0.183 ± 0.008 | 97.62 ± 8.83 | 1.88 ± 0.19 |
‡Kinetic data calculated from eight data points obtained in triplicate by measuring the accumulation of NADH at a wavelength of 340 nm during the oxidation of D‐mannitol over the period of an hour. Assay was performed in 100 µL 50 mM HEPES with 150 mM NaCl (pH 7.4), 1 mg/ml BSA, 1 mM NAD+, and 90‐900 nM pfMDH. Two‐fold serial dilutions were performed on the substrate where the maximum final concentration of D‐mannitol was 200 mM. ND = No detection, detection limit of 0.001 M−1s−1.
Table 2.
Corrected Kinetic Constants for Mutant pfMDH on D‐Arabitol‡
| Enzyme | k cat (s−1) | KM (mM) | k cat/KM (M−1s−1) |
|---|---|---|---|
| E133A | 0.142 ± 0.006 | 80.91 ± 7.94 | 1.76 ± 0.19 |
| E133Q | 0.122 ± 0.004 | 75.93 ± 5.69 | 1.60 ± 0.13 |
| N300A | 0.093 ± 0.004 | 339.27 ± 22.03 | 0.27 ± 0.02 |
‡Kinetic data calculated from eight data points obtained in triplicate by measuring the accumulation of NADH at a wavelength of 340 nm during the oxidation of D‐arabitol over the period of an hour. Assay was performed in 100 µL 50 mM HEPES with 150 mM NaCl (pH 7.4), 1mg/ml BSA, 1 mM NAD+, and 90‐900 nM pfMDH. Two‐fold serial dilutions were performed on the substrate where the maximum final concentration of D‐arabitol was 200 mM. ND = No detection, detection limit of 0.001 M−1s−1.
Table 3.
Corrected Kinetic Constants for Mutant pfMDH on Meso‐Erythritol‡
| Enzyme | k cat (s−1) | KM (M) | k cat/KM (M−1s−1) |
|---|---|---|---|
| E133A | >0.0025 | ≫1.8 | 0.00137 ± 0.00005 |
| E133Q | >0.0023 | ≫1.8 | 0.00126 ± 0.00005 |
| N300A | ND | ND | ND |
‡Kinetic data calculated from eight data points obtained in triplicate by measuring the accumulation of NADH at a wavelength of 340 nm during the oxidation of meso‐erythritol over the period of an hour. Assay was performed in 100 µL 50 mM HEPES with 150 mM NaCl (pH 7.4), 1 mg/ml BSA, 1mM NAD+, and 0.4‐4 µM pfMDH. Two‐fold serial dilutions were performed on the substrate where the maximum final concentration of meso‐erythritol was 1.8M. ND = No detection, detection limit of 0.001 M−1s−1.
Figure 5.
Energetic changes of the measured kinetic constants between WT pfMDH and selected mutants on D‐mannitol (black), D‐arabitol (light gray), and meso‐erythritol (dark gray). Residues are grouped as directly or indirectly involved in catalysis. Mutant labeled “Triple” is the triple mutant containing the H303A, R373A, and K381A mutations. Changes in free energy were calculated using the first order Arrhenius equation (Equation and constants provided in Supplementary Figure S5).