Enzyme Activity Measurement for Galactokinase

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In most organisms, the conversion of β-D-galactose to the more metabolically useful glucose 1-phosphate is accomplished by the action of four enzymes that constitute the Leloir pathway. Galactokinase (EC 2.7.1.6; ATP:alpha-D-galactose 1-phosphotransferase) catalyzes the second step in this pathway, namely the conversion of α-D-galactose to galactose 1-phosphate as indicated in figure 1. Galactokinase has attracted significant research attention for many years due to its important role in sugar metabolism and conversion. For example, defects in this enzyme in human result in the diseased state referred to as galactosemia. Also, most recently it has been developed via directed evolution to synthesize new natural and unnatural sugar-1-phosphates. Additionally, galactokinase-like molecules can serve as the sensors for the intracellular concentration of galactose and, under suitable conditions, function as transcriptional regulators.

Figure 1: The Leloir pathway. The enzymatic steps involved in the conversion of b-D-galactose 1-phosphate.
Reference: Holden H M, et al. Cellular & Molecular Life Sciences Cmls, 2004, 61(19-20):2471-2484.

Galactokinases from various organisms including human, yeast, plants, and bacteria were isolated, expressed, and characterized. These enzymes exhibit distinct specific activities. For instance, galactokinase from Saccharomyces cerevisiae (ScGalK) is over 150 times more efficient than galactokinase from Escherichia coli (EcGalK). Moreover, substrate specificity studies revealed that galactokinase could tolerate modifications of galactose (Gal) at C-2 and C-6. For example, other than Gal, 2-deoxygalactose (Gal2D), and 6-deoxygalactose (Gal6D, also known as D-fucose) are possible substrates of EcGalK and ScGalK. Note that the galactokinases from mammals and from E. coli have no activity with N-acetyl-alpha-D-galactosamine. Probably because of the substrate diversity, the activity assays of galactokinase using spectrophotometry have not been well established in an industrial scale, although activity measurement using other methods was reported before. Creative Enzymes is fully competent to provide the most accurate enzymatic assay for galactokinase by spectrophotometric assays. The activity of galactokinase was measured spectrophotometrically by a coupled assay. The rate of conversion of alpha-D-galactose to alpha-D-galactose 1-phosphate was monitored by coupling the reaction with the formation of lactate from pyruvate in the presence of NADH. Therefore, the activity of galactokinase was determined at 340nm by measuring the formation of NADH.

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Figure 2: The crystal structure of galactokinase from S. cerevisiae.
PDB: 2AJ4