udp - glcua ( ammonium salt ; > 98% purity ) , and nad ( sodium salt ; 98% purity ) were obtained from sigma  aldrich . purified preparations of wild - type hugdh and e161q mutant were obtained using reported procedures . both enzymes were produced as fusion proteins containing an n - terminal extension , which comprised a solubility enhancement tag , a streptavidin tag , and a tobacco etch virus protease cleavage site . the n - terminal extension was then removed , and the enzymes were further purified by gel filtration and anion - exchange chromatography . a 50  mm potassium phosphate buffer at pd 5.9 , 7.0 , 7.9 or 8.8 was used . starting concentrations were 15  mm nad , 2  mm udp - glc and 0.38  m wild - type hugdh or 15  m e161q . after addition of all components , the reactions were incubated at 25  c for 16  h. for determination of the solvent isotope effect , enzymatic conversions in h2o or d2o were performed in a beckman coulter du800 spectrophotometer using a 50  mm potassium phosphate buffer with ph / pd 7.5 . reaction velocity was measured by nadh absorption (   =  340  nm ) . all spectra were recorded on a bruker drx-600 avance spectrometer ( bruker , rheinstetten , germany ) at 600.13  mhz ( h ) . the h nmr spectra were measured at 298.2  k with presaturation ( 1.0  s ) and acquisition of 32k data points . after zero filling to 64k data points , spectra were performed with a range of 7200  hz . chemical shifts were referenced to external acetone ( h  =  2.225  ppm ) . the reactions were directly made in a 5  mm high precision nmr sample tube ( promochem , wesel , germany ) to measure in situ h nmr spectra . samples contained 2  mm udp - glc , 15  mm nad , and 0.084  m wild - type hugdh or 33  m e161q , as well as 50  mm potassium phosphate buffer in d2o ( 0.70  ml , 99.9% d , pd 7.5 or 8.3 ) . 12  h in the magnet by recording up to 64 h nmr spectra in regular intervals . the topspin 3.0 software from bruker was used for processing the nmr spectra after data acquisition . for quantitative analysis of the nmr data , a correction value was subtracted from the integral values of each proton to account for background noise . udp - glcua ( ammonium salt ; > 98% purity ) , and nad ( sodium salt ; 98% purity ) were obtained from sigma  aldrich . purified preparations of wild - type hugdh and e161q mutant were obtained using reported procedures . both enzymes were produced as fusion proteins containing an n - terminal extension , which comprised a solubility enhancement tag , a streptavidin tag , and a tobacco etch virus protease cleavage site . the n - terminal extension was then removed , and the enzymes were further purified by gel filtration and anion - exchange chromatography . full details of the purification protocol are given elsewhere . enzymatic assays were performed in 1  ml of d2o . a 50  mm potassium phosphate buffer at pd 5.9 , 7.0 , 7.9 or 8.8 was used . starting concentrations were 15  mm nad , 2  mm udp - glc and 0.38  m wild - type hugdh or 15  m e161q . after addition of all components , the reactions were incubated at 25  c for 16  h. for determination of the solvent isotope effect , enzymatic conversions in h2o or d2o were performed in a beckman coulter du800 spectrophotometer using a 50  mm potassium phosphate buffer with ph / pd 7.5 . reaction velocity was measured by nadh absorption (   =  340  nm ) . all spectra were recorded on a bruker drx-600 avance spectrometer ( bruker , rheinstetten , germany ) at 600.13  mhz ( h ) . the h nmr spectra were measured at 298.2  k with presaturation ( 1.0  s ) and acquisition of 32k data points . after zero filling to 64k data points , chemical shifts were referenced to external acetone ( h  =  2.225  ppm ) . the reactions were directly made in a 5  mm high precision nmr sample tube ( promochem , wesel , germany ) to measure in situ h nmr spectra . samples contained 2  mm udp - glc , 15  mm nad , and 0.084  m wild - type hugdh or 33  m e161q , as well as 50  mm potassium phosphate buffer in d2o ( 0.70  ml , 99.9% d , pd 7.5 or 8.3 ) . 12  h in the magnet by recording up to 64 h nmr spectra in regular intervals . the topspin 3.0 software from bruker was used for processing the nmr spectra after data acquisition . for quantitative analysis of the nmr data , a correction value was subtracted from the integral values of each proton to account for background noise .
graphical abstract