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BIO 3522-HOMEWORK II (SP 2012)

. 1. Kinetic constants for inhibition studies on enzymes are described below. For each part a-h, determine if the numerical values for the constants are identical or different (increased or decreased). (12 pts) a) Km values in the absence and presence of a competitive inhibitor. b) Vmax values in the absence and presence of a competitive inhibitor. c) Km values in the absence and presence of a noncompetitive inhibitor. d) Vmax values in the absence and presence of a noncompetitive inhibitor. e) Km values in the absence and presence of an uncompetitive inhibitor. f) Vmax values in the absence and presence of an uncompetitive inhibitor. g) How slope would be affected by the presence of noncompetitive inhibitor in a Lineweaver-Burk plot h) How the y-intercept would will be affected by the presence of noncompetitive inhibitor. 2. Using appropriate formula, calculate Go at 25oC and state that reaction is spontaneous,

nonspontaneous or at equilibrium in column 3 of the following table. (show the steps of calculations for at least one Keq). (10 pts)
Keq 75 50 1 104 106 3. a) You have performed a series of experiments determining the Ki values for three competitive inhibitors. The following table lists the results: (5 pts) Inhibitor Ki (M) A 6.5 B 1.5 C 0.25 i) Which inhibitor binds with higher affinity to the free enzyme? Explain. ii) If the same concentration of inhibitor were used in each experiment, which inhibitor would give the smallest value of Km? Explain your answer for proper credit. b) You want to load 10 g of protein in 15 L into one of the 10% polyacrylamide gel well. The protein needs to be in 1X buffer and in a total volume of 0.250 ml. You are given a 5.58 mg/ml protein solution, a 20X sample buffer, and distilled water. How much of each would you mix together to make required volume? Show all steps of calculation. (5 pts) Go'(kJ/mol) Startingwith1Mreactantsand products,thereactionis:

4. The following is an image of Coomassie Blue dye stained SDS-PAGE gel. The details of gel lanes are given below. Excel generated graph and calculations will not be accepted.

-Lane 1 and 7 are low molecular weight protein standards (Phosphorylase b- 97,400, BSA-66,200, Ovalbumin-45,000, Carbonic anhydrase-31,000, Trypsin inhibitor-21,500, and Lysozyme-14,400) and lane 9 is a prestained protein standards. -Lane 3 and 5 are ammonium sulfate fractionated partially purified proteins from eggwhite. -Use Lane 7 and Lane 5 to measure protein migration (use ruler shown on right hand side) and answer questions below: a) How many bands are in the ammonium sulfate partially purified protein? What does this tell you? (2 pts) b) You believe that band shown by an arrow is Ovalbumin. Using low MW standards migration patterns determine MW of this protein. To better explain prepare a Table (as you did in SDS-PAGE lab) and plot (use graph paper). Describe the method how did you obtain the molecular weight? (10 pts) c) Is this molecular weight necessarily the molecular weight of the functional protein? Justify your answer with proper explanation. (3 pts) 5. Calculate vi (initial velocity in the presence of inhibitor) and degree of inhibition caused by a competitive inhibitor under the following conditions: a) Substrate [ ] = 5x103M and b) Inhibitor [ ] = 2x103 M. Assume that Km = 2x103 M, Ki = 1.5x104 M and Vmax = 270 nmoles.L-1.min-1. (10 pts)

6. A research group at UTSA discovers a new enzyme called UTSase that catalyzes the following chemical

reaction: The researchers begin to characterize this enzyme.

(12 pts)

UTSase UTSA SUCCESS

a) In the first experiment, with [ET] at 4 nm they find that the Vmax is 1.6 M s-1. Based on this experiment, what is the kcat for UTSase? (include appropriate units) b) In another experiment. With [ET] at 1 nm and [UTSA] at 30 M, the researchers find that VO = 300 nm s-1. What is the measured Km of UTSase for the substrate UTSA? (include appropriate units) c) Further research shows that the UTSase used in the first experiments was actually contaminated with a reversible inhibitor called LAZINESS at an estimated concentration of 5.5 M. When LAZINESS is carefully removed from the UTSase preparation, the measured Vmax in (a) is 1.6 M s-1 and the Km in both a) and (b) is now 6.5 M. For the inhibitor LAZINESS, determine the type of reversible inhibition and calculate the value of Ki (include appropriate units).
7. Determine the values of Km and Vmax (using Lineweaver-Burk plot) for the decarboxylation reaction of a -keto acid. The data given below: (12 pts). Use graph paper for plot. voIncludethefollowingitemsforpropercredit [S] (molL1) (mMmin1) 2.5 1 0.714 0.526 0.25 0.588 0.5 0.417 0.37 0.256 a)Completedtable b)PlotagraphusingExcelorGraphpaper c)IncludeallcomponentsthatwillbeusedtodetermineKmandVmax.

8. Lysozyme was assayed at bacterial cell wall, [S] = 3.5x10-6 g. The Km for the substrate = 7x10-3g. At the end of one minute 4.0% of the cell wall hydrolyzed. Answer the following questions using this information: (9 points) a) What percent of bacterial cell wall will be hydrolyzed at the end of 3 minutes assuming product formation is in 1st order of reaction? b) What will be a concentration of hydrolyzed product at the end of 4 minutes? c) Determine maximal velocity (Vmax) with the enzyme concentration used. 9. a) The Km of an enzyme of an enzyme-catalyzed reaction is 7.5 M. What substrate concentration will be required to obtain 75% of Vmax for this enzyme? (same enzyme was used in part a and b) (5 points) b) Calculate the Ki for a competitive inhibitor whose concentration is 7.5 x10-6 M. The Km in the presence of inhibitor was found to be 1.25x10-5 M. (5 points)