Studies were carried out to investigate some kinetic properties of alkaline phosphatase (ALP)extracted from Lepus townsendii liver. Incubation of ALP extract with 4- nitrophenylphosphate(4-NPP) formed the basis for determination of enzyme activity. Spectrophotometric method wasused to assay the enzyme activity, and the kinetic constants- maximum enzyme velocity (Vmax)and Michealis-Menten constant (Km) were evaluated.Figure 2 shows the Lineweaver-Burk plot of ALP assay in the presence of inhibitor NaH;PO4 (0)and the control (+) analysis. [I]: concentration of inhibitor = 0.67 mM.1/V,uM*min x 104 40-3020y-Intercept10x-intercept-1/K. (1+ [1]/K)/12 1/[S)x 102(M)Figure2(i)Estimate Km and Vmax in the absence and presence of the inhibitor.(ii)What is the type of inhibition illustrated by the inhibitor? Explain.

Enzymes are protein molecules that increase the rate of reaction by decreasing the activation energy…

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Biochemistry

9th Edition

ISBN:9781319114671

Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.

Chapter1: Biochemistry: An Evolving Science

Section: Chapter Questions

Problem 1P

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To purify a given enzyme from a crude extract that contains it, fractional precipitations, exchange chromatography are successively tested ionic and exclusion chromatography, with the results shown in the following table: Volume of dissolution Protein concentration Enzymatic activity (ml) (mg/ml) (U/ml) a) Calculate the percentage of recovery of the enzyme after each of the manipulations to which it has been subjected. b) Indicate if it is necessary to continue purifying the enzyme or if it is expected to have reached its electrophoretic homogeneity.
Vmax and Km were calculated for enzyme substrate data. Results Y=0,0063x+0,3929 Km =62,4 mikromol/L Vm =159 mikromol/L.min But two additional data pairs were obtained at low concentrations and new graph equation Y=0,0063x+0,391 Recalculate Vmax and Km and compare and comment with previous results.
There are six classes of enzymes, namely oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. (i) (ii) Cell disruption is one of the steps performed during enzyme production. Explain the reason of performing cell disruption in enzyme production and provide THREE (3) enzyme extraction methods for this type of enzyme. Explain the principles of gel electrophoresis as one of the methods used for enzyme purification.
Briefly comment on the differences of using a fixed-time assay versus a kinetic assay to measure enzyme activity. Is it reasonable to assume that the reaction velocity obtained by measuring the amount of product after 30 minutes in a fixed-time assay is directly proportional to absorbance? How could you determine whether this was the case? Word limit 180 words including citation and reference
A purified protease enzyme from the fungus Aspergillus sp. Is tested in a laboratory. This enzyme was lyophilized as a white powder. When reconstituting with phosphate buffer pH 7.2 the active enzyme is obtained. To check its purity, an electrophoresis is performed where a single band of approximately 70,000 molecular weight is observed. The solution with enzymatic activity was stored at 4ºC for later use. A few days later it was found that the enzymatic activity had been lost and in the electrophoretic analysis, instead of a single band there were three bands of weights 40,000, 20,000 and 10,000. Come up with a reasoned explanation of what might have happened to the enzyme
The protein catalase catalyzes the reaction 2H,O,(aq) — 2H,O(l) + O,(g) and has a Michaelis-Menten constant of KM = 25 mM and a turnover number of 4.0 × 107 s¯¹. The total enzyme concentration is 0.010 µM and the initial substrate concentration is 4.83 µM. Catalase has a single active site. Calculate the value of Rmax (often written as Vmax) for this enzyme. Rmax Calculate the initial rate, R (often written as V), of this reaction. R = ×10 mM.s-1 mM-s-1
Consider an enzyme-catalyzed reaction that follows Michaelis-Menten kinetics with KM =3.0 mmol·dm-3. What concentration of a competitive inhibitor with KI = 20 umol·dm-3 willreduce the rate of formation of product by 50% when the substrate concentration is held at 0.10mmol·dm-3.
For an enzyme obeying the Michaelis-Menten equation with Km = 5 µM, kcat = 10 s-¹ and a total enzyme concentration of 1 nM, Calculate Vmax. Calculate the substrate concentration at which v = 0.1 Vmax Calculate the substrate concentration at which v = 0.9 Vmax What fraction of the enzyme is bound to substrate when v = 0.9 Vmax? Sketch a graph showing the Michaelis-Menten plot. Make sure you label the axes on your plot. Label on the graph: i) the point on the graph at which S=Km ii) Vmax At low substrate concentration, v = Keat [Etot] [S] Km Circle on your graph where this equation applies. What name is used to refer to keat/Km? : Calculate keat/Km for this enzyme. How does this value compare with the fastest enzymes?
Phosphoglycerate-mutase catalyses the isomerization of 3-phospho-glycerate to 2-phospho-glycerate. The enzyme has a molecular weight of 28,556 g/mol. In assays using 1 milligram of enzyme per assay the Km for 3-phosphoglycerate was 1.2 x 10^-3M and the Vmax was 840 μmole min^-1. What would be the approximate value of kcat (turnover number or molecular activity) of the enzyme under these conditions?
Phosphoglycerate-mutase catalyses the isomerization of 3-phospho-glycerate to 2-phospho-glycerate. The enzyme has a molecular weight of 28,556 g/mol. In assays using 1 milligram of enzyme per assay the Km for 3-phosphoglycerate was 1.2 x 10^-3M and the Vmax was 840 μmole min^-1 or 14 x 10^-6 moles s^-1. What would be the approximate value of kcat (turnover number or molecular activity) of the enzyme under these conditions?
at what substrate concentration is v= 5.0 mM s^-1 for the enzyme catalyzed hydrolysis of trehalose?
Phosphoglycerate-mutase catalyses the isomerization of 3-phospho-glycerate to 2-phospho-glycerate. The enzyme has a molecular weight of 28,556 g/mol. In assays using 1 milligram of enzyme per assay the Km for 3-phosphoglycerate was 1.2 x 10^-3M and the Vmax was 840 μmole min^-1. What would be the Vmax in units of mole s^-1?

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1/V, uM'min x 10 40 30 20 10 y-Intercept x-intercept