Name___________________________ Section__________________ 7.014 Problem Set 2 Please print out this problem set and record your answers on the printed copy. Answers to this problem set are to be turned in to the box outside 68-120 by 5:00pm on Friday March 2, 2007. Problem sets will not be accepted late. Solutions will be posted online. 1. Lipids (a) Carbohydrates and lipids are both made up of long hydrocarbon chains, but they have very different properties. What is the major distinction between carbohydrates and lipids? (b) Membranes are made up of many components and, in some membranes, phospholipids are the major component. (i) Below is an example of a saturated phospholipid (Where the intersection of two lines represents a carbon atom with hydrogen atoms to fill available covalent bonds). Draw a square around the portion of the molecule that is hydrophobic and a circle around the portion that is hydrophilic. Phospholipids are also sometimes represented like this: 12 Question 1 continued (ii) Using the simplified structure above, draw what a lipid bi-layer looks like in an aqueous environment. Explain what causes the lipids to create this formation. (iii) In your depiction of a lipid bi-layer drawn above, what type of force is acting… - …between the phosphoglycerol (hydrophilic) component and the fatty acid (hydrophobic) component of the phospholipids? - …between the phosphogroup and the surrounding aqueous environment? - …between the fatty acid groups of different phospholipids molecules? (iv) In reality, membranes are composed of several different types of lipids, as well as proteins. One reason why there are multiple types of lipids is to ensure that the membrane remains fluid so that proteins, lipids and small molecules can move through and within the membrane. In particular, there is always a mixture of saturated and unsaturated phospholipids. Give a short explanation of why a membrane containing unsaturated phospholipids would be more fluid than a membrane made exclusively of saturated phospholipids. 2. There are thousands of different kinds of enzymes and each enzyme recognizes a specific substrate or substrates (substrates are the molecules upon which an enzyme acts). You are working in a lab that studies the activity of an enzyme called a nuclease that was purified from the gram-negative bacteria Serratia marcescens. A nuclease is an enzyme that cleaves or breaks apart DNA or RNA by hydrolyzing the phosphate-sugar backbone. The work in your lab has shown that there are four residues important for binding or catalysis of the substrate.3 Question 2 continued (a) Below is the amino acid sequence of the Serratia nuclease. Those amino acids important for binding or catalysis are marked by being enlarged and bolded. These amino acids are Arg78, His110, Asn131 and Glu158. 10 20 30 40 50 60 MRFNNKMLAL AALLFAAQAS ADTLESIDNC AVGCPTGGSS NVSIVRHAYT LNNNSTTKFA 70 80 90 100 110 120 NWVAYHITKD TPASGKTRNW KTDPALNPAD TLAPADYTGA NAALKVDRGH QAPLASLAGV 130 140 150 160 170 180 SDWESLNYLS NITPQKSDLN QGAWARLEDQ ERKLIDRADI SSVYTVTGPL YERDMGKLPG 190 200 210 220 230 240 TQKAHTIPSA YWKVIFINNS PAVNHYAAFL FDQNTPKGAD FCQFRVTVDE IEKRTGLIIW 250 260 AGLPDDVQAS LKSKPGVLPE LMGCKN If the role of this enzyme is to cleave DNA and RNA, why does it make sense that Arginine (R) and Histidine (H) are two of the amino acids important for binding the substrate? (b) As stated above, it is known that these residues are important for binding or catalysis. You want to test for which of these functions (binding or catalysis) the amino acids Arg78 and His 110 is important. To perform this test you change Arg78 and His110 to different amino acids and then monitor if the nuclease can still cleave DNA. Below is the outline for the assay: 1. Incubate either or wild-type (wt) or mutant (mt) enzyme with DNA. 2. After several minutes, you isolate the DNA from the reaction. 3. Run the DNA pieces through an agarose gel matrix (see the Research Method box on pg. 319 in Purves et. al.) using an electric current. At this time it is not important to understand the technique, but it is important to understand that the electric current causes DNA fragments of different sizes to separate from each other and that this separation can be visualized. The chart below shows which enzymes you will use in the assay: Name of Enzyme Amino Acid Position of Amino Acid wt* Arg 78 mt1 Ala 78 mt2 Lys 78 mt3 Trp 78 wt* His 110 mt4 Ala 110 mt5 Lys 110 mt6 Trp 110 * note that there is only one “wt” enzyme. “wt” is listed one time for each of the different amino acids you are studying.Question 2 continued Below is a representation of your agarose gel after you have separated the DNA. There are multiple lanes on the gel. Each lane contains the DNA from one of your different incubations. The version of the enzyme (wt or mt) that was incubated with the DNA in a specific lane is noted above the lane (no = no enzyme added to that DNA). wt no mt1 mt2 mt3 mt4 mt5 mt6 big DNA small DNA (i) Based on your results above do you think that Arg88 is more likely to be important for binding the DNA or for cleaving the DNA? Why? (ii) Based on your results above do you think that His110 is more likely to be important for binding the DNA or for cleaving the DNA? Why? (c) Your lab also is interested in understanding how much energy is required to cleave the phosphodiester bond. To do this, you perform an assay similar to the one described above, but this time you allow the reaction to reach equilibrium. You then measure the amount of full-length DNA you started with and the amount of cleaved DNA after the reaction reaches equilibrium. The reaction can be described using the following equation: 1 full length DNA molecule 2 shorter DNA molecules or A 2B 4Question 2 continued The final concentrations of your reactants and products are: A = 3 nM B = 4.5 mM (i) Based on these measurements, determine the amount of standard free energy (ΔG◦´) for A 2B. Show your work. (ii) What does your answer tell you about how the reaction proceeds under standard biological conditions? (iii) Why
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