The kcat and KM for chymotrypsin-catalysed cleavage of a synthetic substrate, S, were determined to be 60 s-1 and 0.5 mM, respectively, using steady-state kinetics. The concentration of enzyme in the assay was 5 x 10-8 M.  Sketch a graph on the axes shown in Figure 1 below showing how the initial rate of the reaction varies with [S], making use of the values for KM and Vmax. Label the axes with appropriate titles and units.

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)

Calculate the mass of invertase (in mg) and  concentration of invertase (in mM) contained in a 25.0mL sample of yeast extract that has 3,000 total units of activity, assuming that pure invertase has a specific activity of 1,000 units/mg with a mass of 270kD.

A16 Compound A is the substrate for two enzymes, E, and E, involved in the synthe- sis of an amino acid and an antibiotic, respectively. Their reaction rates, r, and r2, at an arbitrary concentration of E, and E, were measured at different concen- trations of A. However, the data were not labeled properly (shown in Table P.4.1), and the student who did the measurement needs help to sort out which dataset is for E,. Determine the K and rmax for both enzymes, with respect to the concen- tration of A. Which set of data is more likely to be for E, and which for E,, and why? Table P.4.1 Reaction kinetics of a common substrate A for two enzymes. Concentration of A (mM) 0.2 0.6 1.2 2 4 6. 8 9 12 15 Reaction rate (r,) (mmol/L'min) Reaction rate (r.) (mmol/L*min) 3.33 4.29 4.62 4.76 4.84 4.88 4.9 4.92 4.94 4.95 4.96 4.97 0.09 0.23 0.38 0.5 0.6 0.67 0.71 0.75 0.8 0.82 0.86 0.80

The enzyme glucose oxidase isolated from the mold Penicillium notatum catalyzes the oxidation of B-D-glucose to D-glucono-6-lactone. The enzyme is highly specific for the B anomer of glucose and does not affect the a anomer. In spite of this specificity, the reaction catalyzed by glucose oxidase is commonly used in a clinical assay for total blood glucose -that is, for solutions consisting of a mixture of B- and a-D-glucose, as well as other sugars present in blood. The oxidation proceeds in the presence of oxygen and forms hydrogen peroxide, in addition to the lactone. A second enzyme, called peroxidase, catalyzes the reaction of hydrogen peroxide with colorless compounds to create a colored product, which is quantified with a simple spectrophotometer. What are the circumstances required to make this possible? Aside from allowing the detection of smaller quantities of glucose, what advantage does glucose oxidase offer over the Fehling's reagent for measuring blood glucose?

*The enzyme glucose oxidase isolated from the mold Penicillium notatum catalyzes the oxidation of 3-D-glucose to D-glucono-6- lactose. This enzyme is highly specific for the ß anomer of In spite of this glucose and does not affect the a anomer. specificity, the reaction catalyzed by glucose oxidase is commonly used in a clinical assay for total blood glucose that is, for solutions consisting of a mixture of 3- and a-D- glucose. What are the circumstances required to make this possible? Aside from allowing the detection of smaller quan- tities of glucose, what advantage does glucose oxidase offer over non-enzymatic oxidizing agents like Tollens reagent? *Is B-D-glucosamine a reducing sugar?

The enzyme glucose oxidase isolated from the mold Penicillium notatum catalyzes the oxidation of β-Dglucose to D-glucono-δ-lactone. This enzyme is highly specific for the β anomer of glucose and does not affect the α anomer. In spite of this specificity, the reaction catalyzed byglucose oxidase is commonly used in a clinical assay for total blood glucose—that is, for solutions consisting of a mixture of β- and α-D-glucose. What are the circumstances required to make this possible? Aside from allowing the detection of smaller quantities of glucose, what advantage does glucose oxidase offer over Fehling’s reagent for measuring blood glucose?

During the early stages of an enzyme purification protocol, when cells have been lysed but cytosolic components have not been separated, the reaction velocity-versus-substrate concentration is sigmoidal. As you continue to purify the enzyme, the curve shifts to the right. Explain your results. This is an allosteric enzyme and you must use a Lineweaver-Burk plot to determine KM and Vmax correctly. This is an enzyme that displays Michaelis-Menten kinetics and you purify away an inhibitor. This is an allosteric enzyme and during purification you purify away an activator. This is an allosteric enzyme displaying a double-displacement mechanism and during purification you purify away one of the substrates: This is an enzyme that displays Michaelis-Menten kinetics, and you must use a Lineweaver-Burk plot to determine KM and Vmax correctly.

The enzymatic activity of PFK1 is generally measured by set- ting up a coupled enzyme assay system whereby aldolase, triose phos- phate isomerase, and glycerol-3-phosphate dehydrogenase are added to the assay mixture. For the latter enzyme, NADH is added and its change in concentration is readily monitored at 340 nm. Write the chain of reactions catalyzed by these enzymes using structural formulas, label substrates and products, and explain why the coupled en- zyme assay system leads to oxidation of NADH. While the chain of reac- tions is similar to those in glycolysis, there is a critical difference because of the dehydrogenase enzyme. Describe how this enzyme causes the chain of reactions to differ from those in glycolysis.

A16 Compound A is the substrate for two enzymes, E, and E, involved in the synthe- sis of an amino acid and an antibiotic, respectively. Their reaction rates, r, and r, at an arbitrary concentration of E, and E, were measured at different concen- trations of A. However, the data were not labeled properly (shown in Table P.4.1), and the student who did the measurement needs help to sort out which dataset is for E,. Determine the K, and rmax tration of A. Which set of data is more likely to be for E, and which for E,, and why? for both enzymes, with respect to the concen- Table P.4.1 Reaction kinetics of a common substrate A for two enzymes. Concentration of 0.2 0.6 1.2 3 4 6. 8 9. 12 15 A (mM) Reaction rate (r.) (mmol/L'min) 3.33 4.29 4.62 4.76 4.84 .88 4.9 4.92 4.94 4.95 4.96 4.97 Reaction rate (r,) (mmol/L*min) 0.09 0.23 0.38 0.5 0.6 0.67 0.71 0.75 0.8 0.82 0.86 0.80