1. The mechanism shown below is proposed for the reversible NAD+-dependent oxidation of lactate to
pyruvate by lactate dehydrogenase. Answer the questions asked below.
(a) What is the role of His 195 in this mechanism?
(b) What is the role of Arg 171 in this mechanism?
(c) What is the role of NAD+ in this mechanism?
(d) Why would the oxidation not occur without NAD+?
(e) Redraw the mechanism for the reduction of pyruvate to lactate.
2. In 1969, William Jencks of the University of Wisconsin published his famous treatise on enzymology, “Catalysis in Chemistry & Enzymology.” In this publication, Dr. Jencks describes four major factors that contribute to the ability of enzymes to catalyze reactions. What are these four factors? Published data indicate that the hydrolysis of acetylsalicylic acid is about 100 times faster that the hydrolysis of phenyl acetate (reactions shown below). How does the work of Jencks apply?
3. The first step of glycolysis in most organisms is the phosphorylation of glucose by the ATP-dependent enzyme Hexokinase. The reaction is shown below.
ATP is a substrate for Hexokinase. However, at high levels of intra-cellular ATP, Hexokinase slows down. Explain how this is possible.
4. From the plot of initial velocity (V) versus concentration of substrate below, obtain or calculate the parameters listed. The enzyme concentration in the reaction mixture is 0.0010 umol/L. The x-axis (independent variable) is [S] multiplied x 1000. In other words, 1.0 on the x-axis means 1.0 x 10-3 M. This is done to make the plotting easier. Show the proper units on Km, Vmax, Kcat and Kcat/Vmax. This is not an LB plot, but you can obtain the information regardless. Check in your text and the enzyme notes I posted on the ECN. They will help you. SHOW ALL calculations and units cleKm: ____________
kcat / Km: ___________.
5. The Lineweaver-Burke plot shown below is for the oxidation of malate to oxaloacetate in the TCA cycle
in mitochondria which is catalyzed by malate dehydrogenase:
Calculate the values of Km and Vmax for malate dehydrogenase from the LB plot shown below. The
original values of V has units of umol/min and [malate] is expressed in mM.
On a separate sheet of paper, draw a reasonable mechanism by which this oxidation is happening.
6. Write out the Michaelis-Menten equation and show that when V0 = 0.5Vmax, [S] = Km.
7. Using your understanding of inhibition mechanisms, explain the following observations:
a) For reversible competitive inhibition, the measured value of Km will increase as [ I ] increases, but the value of Vmax remains constant as [ I ] increases.
b) For reversible non-competitive inhibition, the measured value of Km remains constant as [ I ] increases, but the value of Vmax decreases as [ I ] increases.
8. Salicylate inhibits the catalytic activity of glutamate dehydrogenase, an NADH-dependent enzyme responsible for the oxidative deamination of glutamate to -ketoglutarate in mitochondria. Given the following data, construct a Lineweaver-Burke plot. What kind of inhibition does salicylate exhibit? Calculate Km and Vmax for the uninhibited reaction. Assume that the concentration of the enzyme is held constant. (10 points) do this on excel. Show all calculations and units.
The Structural formulas of Glutamate and Salicylate are shown below. Given these formulas, explain the type of inhibition observed.
9. Carbonic Anhydrase is strongly inhibited by the drug acetazolamide which is used as a diuretic (increases the production of urine to help decrease blood pressure) and to treat glaucoma (to help reduce fluid pressure in the eye). Carbonic anhydrase plays an important role in these and other secretory processes because it participates in regulating the pH and bicarbonate content of a number of body fluids. The experimental curve of initial reaction velocity (as a % Vmax) versus [S] for the carbonic anhydrase reaction is illustrated below on the upper curve with no inhibitor present. When the experiment is repeated in the presence of acetazolamide, the lower curve is obtained. From an inspection of the curves and your knowledge of the kinetic properties of competitive, non-competitive and mixed enzyme inhibitors, determine the nature of the inhibition present and explain what is happening on the enzyme.
10. The following plot was obtained for an enzyme with substrate and separately in the presence of a ligand which was a suspected inhibitor. The line with the black circles was obtained for the enzyme and substrate with no ligand present. The line with the black triangles was obtained for the exact same experiment except that a fixed amount of the ligand was also present with the substrate.
a) explain the shape of the line obtained when the substrate was with the enzyme alone.
b) explain the change observed when the ligand was added to the system.
c) what do these data indicate about the ligand? Explain