X Ter 1 7°C Cloudy 6 OF 16 QUESTIONS REMAINING The rate of an enzyme catalysed reaction was measured with increasing substrate concentrations in the presence of 0 μM, 2 μM or 5 μM of a non-reactive substrate analogue capable of binding to the active site. The following double reciprocal Lineweaver-Burk plot was produced from the data obtained. Match each of the following terms with the correct label (A-G) on the plot. Prompts 11/[5] (B) (A) (c) (D) (E) (G) (F) Answers Select match Select match Reaction with 2 μM of the substrate analogue (3) 1/KM Last saved 10:10:33 Questions Filter (16) ▼ Q Search Select match K First < Previous Next > Last > BB •

Discuss how the equation for enzymatic reaction (given below) was demonstrated in the experimental results in Table 1. Use the appropriate color codes (-), (+), (++), (+++) to describe the expected results in Table 1 Table 1. Enzyme Action MIXTURE ENZYME ACTIVITY Water + catechol (tt A)   Enzyme + catechol (tt B)   Water + enzyme (tt C)

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You will perform the protocol below for the calf intestinal alkaline phosphatase (CIP) provided. For each reaction, your final enzyme concentration should be 10 nM CIP. Note: Enzymes purchased are typically labelled with their “units of activity” (U), as this relates to how much enzyme is needed to catalyze a reaction. The 100 nM CIP provided has approximately 3 U/mL and was diluted 1 in 1,000 from a 500 U/mL purchased enzyme. 1) Create a table (similar to the one below) to help you determine and keep track of what to add to each of the cuvettes in which your reactions will be measured. The five different concentrations of PNPP should be: 25, 50, 100, 200, 300 μM. Each reaction will be in a final volume of 1 mL and contain 10 nM alkaline phosphatase. Concentrations of stock solutions: 1.0 mM PNPP, 100 nM calf intestinal phosphatase

A synthetic substrate, the para-nitrophenylacetate (PNPA), is used to monitor enzyme activity of protein P. The product of the hydrolysis reaction absorbs at 410 nm with a molar extinction coefficient of 4 000 M-¹.cm-¹. 1- Write the reaction catalyzed by the protease P using the pNPA substrate. 2- The enzyme extract is too concentrated and a 1/300 dilution is needed for enzyme tests. Considering that you would need at least 600 µL of diluted enzyme extract for activity tests, indicate which volume of buffer and enzyme extract you must use for the dilution.

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

WMapp E+S ES E+P Vmax Km =Vmax 2 -1 a = 1+ [1]/K, X intercept = Kcat [E]T Km Question 6 In the simple enzyme-catalyzed reaction below, which of the rate constants would be second-order? E+S ES →E+P k-1 O k2 O k-1 O k1

The accompanying figure shows three Lineweaver–Burk plots for enzymereactions that have been carried out in the presence, or absence, of aninhibitor. Indicate what type of inhibition is predicted based on eachLineweaver–Burk plot. For each plot indicate which line corresponds to thereaction without inhibitor and which line corresponds to the reaction withinhibitor present.

At what substrate concentration would an enzyme with a kcat of 25.0 s-1 and a KM of 3.5 mM operate at 25% of its maximal rate? How many reactions would the enzyme catalyze in 45 seconds when it is fully saturated with substate, assuming the enzyme has one active site?

Staphylococcal nuclease has a ΔΔG‡ of -84.1 kJ mol-1 at 25.0 °C. If the uncatalyzed rate is 0.630x10-13 µmol s-1, calculate the enzyme-catalyzed rate in µmol s-1. (Use R = 8.3145 J mol-1 K-1)