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1. Enzyme Z catalyzes the reaction: A NAD <> B NADH H' Besides following the [H' using a pH meter. the reaction can be monitored spectroscopically at 340 nm Thisi: the wavelength at which NADH absorbs light Some of the first enzymes that were worked on in biochemistry were chydrogenases, like our enzyme Z. because of this advantage Radioisotopes were not yet available a) Diagram what the kinetic assay results would br if the reaction was measured from left to right. What would be if the assay was measured from right toleft? b) Describe how you would accurately determine enzyme Z's Km for NAD Describe the experimental set-up and the analysis of data. c) Imagine that twenty I.U.) of enzyme Z were catalyzing the above reaction for one minute, under Vmas conditions, in 3.00 ml assay volume. The assay buffered with 20 mM phosphate buffer, pH 7.60. What will the pH be at the end of that one minute? 2. You are going to measure the enzyme activity of enzyme "T". Enzyme "T" catalyzes the reaction: S +> P. Enzyme "T" has Kmof 1.0 : 10° M for S. You mix the following solutions together: 2.60 r ml 10 mM phosphate buffer, pH 7.2 0.30 ml 30 mM S 0.10 ml enzyme " The results of this enzyme assay are plotted below: Time (seconds) a) What is Vo for the above reaction? b) How many International Units (I. U.'s) of enzyme "T" were present in the assay? c) Carefully sketch in, on the above graph, the expected initial velocity if the substrate concentration had initially been 1.5 x 10°M. d) Let assume that .0x 10-1 mol of enzyme" was present in both assays-i.e. in part "a" and in part "c". Calculate the turnover number for enzyme "T" 2of3 3. The following table lists properties of four different proteins: Protein pI MW chymotrypsinogen 9.4 23,200 hemoglobin 7.0 64.500 lysozyme 11.1 14.100 ovalbumin 4.9 45,000 a) What the order of elution of the above proteins from sizing column at pH 7? b) What the order of elution from an anion exchange column at pH 7? c) What the order of migration of the above proteins in SDS-PAGE gel? d) How will these proteins band in an agarose IEF gel? 4. Part of the H' portion of the Bohr effect occurs at histidine 146 at the C- -termini of the ß-subunits in hemoglobin. In hemoglobin Hiroshima, that histidine has been changed to an aspartic acid residue. a) How will hemoglobin Hiroshima migrate (band), compared to normal hemoglobin, in a native PAGE gel? Why? b) How will hemoglobin Hiroshima migrate (band), compared to normal hemoglobin, in an SDS- PAGE gel? Why c) How will hemoglobin Hiroshima migrate (band), compared to normal hemoglobin, in an isoelectric focusing gel? Explain. 5. Calculate the missing values (X) in the following enzyme purification table: Volume [Protein] Total Activity Specific Fold Fraction (ml) (mg/ml) I.U./ml LU Yield (%) activity Purification I. crude 1000 10 2 2000 100 X1 1.00 2. resuspended 200 20 12 X2 120 X3 X4 40% NH4)2SO4 pellet 3 affinity column 2 Xs 200 400 X6 200 x, How do you explain the value you calculate for X2(i.e. why is it greater than 2000 I.U.)? 6. A plant is growing in the jungles of South America Would the fatty acid composition of its membrane lipids contain more 18:3 (9,12,15 or more 3:02 Explain 3of3 7. What is this all about? Image from Voet and Voet, Biochemistry, 3rd ed., c2004

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