Problem Set 4-3 PDF

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Problem Set 4 – Enzyme Kinetics, Catalysis and Mechanisms Please note: graph paper has been provided on the last page. Please print as many copies as you desire!! 1. Enzyme kinetics –part 1 The following data has been readapted from a 2000 article published in the journal, Proceedings of the National Academy of Sciences. In part, It reported on the development of an inhibitor for a type of enzyme known as fatty acid hydrolase. In the report, the inhibitor is simply referred to as 53. Below, pertinent data has been taken from the report and readapted for the problem below. Enzyme kinetics data with NO INHIBITOR [S] (M) 0 12.5 25.0 50.0

Vo (M) Min 0 0.435 0.588 0.714

Enzyme kinetics data with 0.002 M of the 53 INHIBITOR [S] (M) 0 12.5 25.0 50.0

Vo (M) Min 0 0.238 0.357 0.500

Please do the following and make use of graph paper and make sure to properly label both axes a. In part a, please use both the no inhibitor and the inhibitor data and draw/sketch a Michaelis– Menten plot. Please use the same plot for both sets of data ( no inhibitor and inhibitor) b. In part b, please use the same plot for both sets of data (no inhibitor and inhibitor) and draw a Lineweaver–Burk (or a double–reciprocal) plot c. From the Lineweaver-Burk plot constructed in part b, estimate the Km value in the presence and absence of inhibitor (please make sure to report proper units) d. From the Lineweaver-Burk plot, estimate the V max value for this reaction for both the +inhibitor data as well as for the no inhibitor data. e. Based on your Lineweaver-Burk plot, the 53 molecule is what type of an inhibitor? And hence, what type of inhibition is displayed? Please briefly explain your response 2. Enzyme kinetics –part 2 The following data has been readapted to fit the purpose of this problem from the journal, Scientific Reports (Nature Publishing Group). It reported on the use of an inhibitor for the enzyme, butyryl cholinesterase.

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The following was the data readapted for the no inhibitor set [S] (mM) 0 0.167 0.200 0.250 0.333 0.500 1

Vo (no units were reported in paper, please keep it the same way) 0 143 154 167 182 200 250

The following was the data readapted and it is for the plus inhibitor set [S] (mM) 0 0.167 0.200 0.250 0.333 0.500 1

Vo (no units were reported in paper, please keep it the same way) 0 33.3 37.0 45.5 52.6 71.4 90.9

Please do the following and make use of graph paper and make sure to properly label both axes a. Using the same plot for both sets of data (no inhibitor and inhibitor) and draw a Lineweaver–Burk (or a double–reciprocal) plot b. From the Lineweaver-Burk plot constructed in part a, estimate the Km value in the presence and absence of inhibitor (please make sure to report proper units) c. Based on your Lineweaver-Burk plot, what type of an inhibitor was designed and tested for the cholinesterase enzyme? Please briefly explain your response d. From the Lineweaver-Burk plot, estimate the V max value for this reaction for both the +inhibitor data as well as for the no inhibitor data. nd

3. In the October 2 , 2012 issue of the journal, EMBO Molecular Medicine, a study was published reporting on the development of an inhibitor for an enzyme present in Mycobacterium tuberculosis, the causative agent of the respiratory/lung ailment tuberculosis. The researchers targeted an enzyme known as InhA with an inhibitor known as pyridomycin. The following enzyme kinetics data has been utilized and readapted from their study to fit the purpose of this question Readapted No Inhibitor data [S] (M)

Vo (they did not report explicit units in their paper, please leave units blank)

0 10 12.5 16.7 25 50

0 1 1.2 1.33 1.54 1.82

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Readapted plus Inhibitor data [S] (M) 0 10 12.5 16.7 25 50

Vo (they did not report explicit units in their paper, please leave units blank) 0 0.42 0.56 0.71 0.91 1.33

a. Using the same plot for both sets of data (no inhibitor and inhibitor) and draw a Lineweaver– Burk (or a double–reciprocal) plot b. From the Lineweaver-Burk plot constructed in part a, estimate the Km value in the presence and absence of inhibitor (please make sure to report proper units) c. Based on your Lineweaver-Burk plot, what type of an inhibitor is pyridomycin that was designed and tested for the InhA enzyme? Please briefly explain your response. d. From the Lineweaver-Burk plot, estimate the V max value for this reaction for both the +inhibitor data as well as for the no inhibitor data. 4. Enzyme catalysis mechanisms The following figure is taken from a paper reporting on the structure published in the journal, Proceedings of the National Academies of Sciences (December 17th, 2013 issue). The study sheds light on an enzyme known to be defective in Krabbe’s disease. Below, a readapted figure has been shown to facilitate the answering of the respective questions. Carefully analyze the reaction mechanism shown below and please answer the following questions.

Please note: the substrate is a sugar molecule undergoing a glycosidic bond cleavage and it is color-coded differently in each section (A= green, B= orange, C= red). a. What are the identities of the catalytic residues? Please provide complete name 3

b. What type of catalysis is shown in part A of the reaction? Please make sure to answer the question with respect to the top and bottom catalytic residue and be certain to mention the catalytic mechanism likely being utilized in that particular step. c. What catalytic mechanism is being utilized in part B? d. What is the purpose/function of the water molecule in part B? 5. Enzyme catalytic mechanism – part II Carefully analyze the catalytic mechanism seen below. This is a catalytic mechanism for a class of proteases known as rhomboid proteases

6. Enzyme Kinetics th In the May 16 , 2006 issue of the journal, The Proceedings of the National Academy of Sciences, researchers reported on the structure of an enzyme known as farnesyl pyrophosphate synthase or FPPS in its abbreviated form. In addition to reporting on the structure, the investigators also performed rigorous enzyme kinetics analysis in the presence of an inhibitor known as risedronate. Specifically, they tested risedronate’s inhibitory abilities on two substrates, geranyl pyrophosphate (GPP) and isopentenyl pyrophosphate (IPP). The data for one of the substrates, IPP, has been readapted to fit the purpose of this question.

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No inhibitor data, S= IPP [S] (M) Vo 0.2 0.29 0.5 1 2

0.005 0.0067 0.01 0.017 0.018

+ Inhibitor (50 nM risedronate) [S] (M)

Vo

0.2 0.29 0.5 1 2

0.0031 0.0045 0.006 0.008 0.01

Please do the following: a. On the same piece of graph paper, plot a Lineweaver-Burk plot for the “no inhibitor” and the “+ inhibitor” data set. Please make certain to label your axes! b. Second, determine the Km and Vmax values for each data set from your graph c. Third, based on your graph, what type of inhibitor is risderonate? 7. Consider the catalytic amino acid residues that comprise the active site of serine proteases. Which three residues are known to comprise this active site? a. Draw their structures (need to only show side chains) closely mimicking as they would appear in an active site. b. Second, show/draw the mechanism of how a serine residue is activated into an alkoxide by a neighboring histidine residue c. Third, show/draw the mechanism of how the activated serine residue would make a nucleophilic attack on a substrate peptide molecule. You can generalize the peptide molecule for ANY serine protease. In other words, you need not draw a peptide bond matching any of the main three serine proteases: trypsin, chymotrypsin and/or elastase. 8. Consider the enzyme  -galactocerebrosidase or GALC whose structure as well as enzyme kinetics and catalytic mechanism were published in PNAS The following data was reported and subsequently utilized [S] (mM)

Vo (M) Min

1 3 7 12 25 50

6 10 14 16 19 21

a. Plot a Michaelis-Menten plot of the above data and estimate the Vmax of the reaction. b. What would be the k cat value of the above reaction if the researchers used a GALC enzyme with a concentration of 0.00987 M. Please make sure to report consistent units

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9. The following enzyme kinetics data was reported in the journal, Nature Chemical Biology (July th 20 , 2014) and it pertains to an enzyme known as butelase whose function is to create peptide rings. The equations below are provided for your reference, however, please be advised that they will NOT be provided on an exam. Thus, you should learn these! kcat = Vmax Catalytic efficiency = kcat [E] Km For parts (a), (b), and (c), please do the following:  Estimate the Vmax  Determine the k cat  Determine the catalytic efficiency  Please note that for (a) and (b), [enzyme] = 0.125 M and for (c), [enzyme] = 0.005 M.

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