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Since only studies on human enzyme are likely to be of value …


Biology Articles » Biochemistry » Sugar recognition by human galactokinase » Methods

Methods
- Sugar recognition by human galactokinase

Protein expression and purification

Human galactokinase was expressed in E. coli as a His6-fusion protein and purified from this source by nickel agarose chromatography as described previously [12]. Point mutations were introduced in to the coding sequence using the QuikChange method [23] and the mutant proteins expressed and purified using the same protocol. The full DNA sequence of each mutant galactokinase was verified (DNA Sequencing Service, Department of Medicine, University of Manchester). Protein concentrations were estimated by the method of Bradford [24].

Galactokinase kinetics

The kinetics of the galactokinase reaction was measured by coupling the production of ADP to the reactions of pyruvate kinase and lactate dehydrogenase at 37°C in a total reaction volume of 150 μl [12,13]. The decrease in A340nm, which results from the oxidation of NADH, was measured in a Multiskan Ascent micro-titre plate reader. The reaction mix contained 20 mM HEPES-OH pH 8.0, 150 mM NaCl, 5 mM MgCl2, 1 mM KCl, 10%(v/v) glycerol, 1 mM NADH, 1 mM DTT, 400 μM PEP, 7.5 U pyruvate kinase (Sigma) and 10 U lactate dehydrogenase (Sigma). All sugars were purchased from Sigma-Aldrich. Reactions were initiated by the addition of enzyme.

It has previously been established that human galactokinase follows an ordered, ternary complex mechanism [12] for which the rate equation is given by v = (kcat. [E]0. [gal]. [ATP])/(KI,ATP.Km,gal + Km,gal. [ATP] + Km,ATP. [gal] + [ATP]. [gal]) where [gal] and [ATP] are the concentrations of galactose and ATP respectively, KI,ATP is a constant relating to the dissociation of the enzyme-ATP complex and Km,gal and Km,ATP are the Michaelis constants for galactose and ATP respectively. At any constant value of [gal] this simplifies to v = kcat,app. [E]0. [ATP]/(Km,ATP,app + [ATP]) where kcat,app = kcat. [gal]/(Km,gal + [gal]). A similar situation holds when [ATP] is held constant [12]. A 5 × 5 concentration grid with a range of sub-saturating constant concentrations of ATP and galactose was set up. Each row or column gave a set of rates at a constant concentration of one substrate. Using these data, kcat,app for each value of [galactose] and [ATP] and the associated standard error were calculated using non-linear curve fitting [25]. Secondary plots were then constructed in which these kcat,app values were plotted against the relevant substrate concentration. Non-linear curve fitting [25] was then used to derive the absolute kcat, Km and kcat/Km values and their associated standard errors. This method has the advantage of avoiding using "saturating" concentrations of substrate which may not be truly saturating, may not be easy to obtain experimentally and may result in unwanted substrate inhibition [26].


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