Page 3 2) 7. J a) Glucosidase I catalyzes hydrolysis of specific glucosidase I is a synthetic trisaccharide, glucose-al-2- oligosaccharides containing glucose. obtained using this trisaccharide as substrate in glucose-al-3-glucose-a-O(CH₂) COOCH,. absence (x-x) and presence of the inhibitor 1- deoxynorjirimycin at concentrations of 50 μM (-), (0-0), and 200 μM (A-A) were used to prepare the Kinetic measurements the Lineweaver-Burk plot below: 1.5 1.0 0.5 0.0 -1.0 0.0 One substrate for 1.0 2.0 100 }M 1/Trisaccharide (mM)-! Estimate the values for Vmax and KM for the trisaccharide substrate in the absence of the inhibitor. Determine whether inhibition by 1-deoxynorjirimycin is competitive, non-competitive or neither.
Parameters such as Km and vmax are used for comparing enzyme activities. If we know the initial rate of reaction and substrate concentration, Km and vmax can be calculated from the Michaelis Menton equation ;
vo = (vmax · [S]) / (Km +[S])
Where, vo is the rate of formation of product, [S] is the substrate concentration, Km is called the Michaelis-Menton constant which represents the enzyme's affinity for the substrate and vmax is the maximum rate at which the enzyme can catalyze the reaction.
vo and [S] can be estimated experimentally but Km and vmax are estimated from the Michaelis Menton plot.
Km = [S] when vo= vmax/2
When we plot vo vs [S] data on a graph, the result is a curve. Accurate values of Km and vmax cannot be determined from a curve so we transform the Michaelis Menton Equation into Lineweaver Burk Equation by taking the reciprocal of vo and [S] and plot a 1/vo vs 1/[S] which gives us a straight line.
Michaelis Menton equation vo = (vmax · [S]) / (Km +[S]) becomes 1/vo = Km/vmax · 1/[S] + 1/vmax
Lineweaver Burk equation : 1/vo = Km/vmax · 1/[S] + 1/vmax
When we plot 1/Vo on the y-axis vs 1/[S] on the x-axis, the plot is called a Lineweaver Burk Plot.
This makes the Lineweaver Burk equation comparable to the straight line y = mx + c equation
where m (the slope of the line) is equal to the ratio Km/vmax while the y-intercept c is equal to 1/vmax.
Lineweaver Burk Plot enables us to more accurately determine the Km and Vmax values.
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