Studies on the substrate specificity of Escherichia coli galactokinase

Organic Letters

Volumn 5, Issue 13, 2003, Pages 2223-2226

  Yang, Jie ^a   Fu, Xun ^a   Jia, Qiang ^b   Shen, Jie ^b   Biggins, John B ^a   Jiang, Jiqing ^a   Zhao, Jingjing ^a   Schmidt, Joshua J ^a   Wang, Peng G ^b   Thorson, Jon S ^a  

^a UNIVERSITY OF WISCONSIN MADISON   (United States)

^b WAYNE STATE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BACTERIAL ENZYME; CARBOHYDRATE DERIVATIVE; GALACTOKINASE;

ARTICLE; CARBOHYDRATE SYNTHESIS; COLORIMETRY; ENZYME ASSAY; ENZYME KINETICS; ENZYME SPECIFICITY; ENZYME SUBSTRATE; HIGH PERFORMANCE LIQUID CHROMATOGRAPHY; IN VITRO STUDY; LIQUID CHROMATOGRAPHY; MASS SPECTROMETRY; NONHUMAN; THIN LAYER CHROMATOGRAPHY;

COLORIMETRY; ESCHERICHIA COLI; GALACTOKINASE; GALACTOSE; GLYCOSIDES; KINETICS; SALICYLIC ACIDS; SUBSTRATE SPECIFICITY;

EID: 0141518596     PISSN: 15237060     EISSN: None     Source Type: Journal    
DOI: 10.1021/ol034642d     Document Type: Article

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34. 2). The bound recombinant proteins were eluted by elution buffer (200 mM imidazole in sodium phosphate buffer). The active fractions were concentrated and further resolved via gel filtration chromatography. The fractions containing homogeneous GalK were collected and concentrated, and the final concentration of protein was determined by Bio-Rad protein assay.
35. 2 (5 mM) in 50 mM sodium phosphate buffer (pH = 7.5) was incubated at 30°C for 5 min, and then GalK (final concentration is 5.6 μM) was added to the reaction system. At various time points, aliquots were analyzed via chromatography (ref 9) and the DNS assay (ref 10).
36. f values of galactose, ATP, and galactose-1-phosphate were 0.51, 0.12, and 0.28, respectively. LC-MS analysis was also consistent with the formation of Gal-1-P.
37. During the reaction process, 50 μL of reaction solution was taken out periodically and transferred to 100 μL of DNS reagent solution (45mM) to assess the reaction progress. Each DNS reaction solution was placed in a bath of boiling water for 15 min and subsequently cooled to room temperature followed by the addition of 15 μL of a 40% potassium sodium tartrate solution to stabilize the color. The absorbance at 575 nm was recorded, and a plot of change in absorbance at 575 nm as a function of time was obtained.
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