PSBIOCH

Assignment 3: Enzymes

Term 3 AY 2015 2016

WRITE YOUR ANSWERS ON SHORT BOND PAPER. GRAPHS MUST BE DRAWN ON GRAPHING PAPER (OR
PRINTED FROM AN EXCEL FILE)
1. The following values were obtained for an enzyme in the presence of 3 different substrates.
Total enzyme concentration [E]o = 1.0 mM; Substrate concentration [S] = 1.0 mM
Substrate
D-glucose
D-fructose
D-mannose

Km (mM)
0.0080
2.0
0.0050

Vmax

kcat (mM/min)

1.0
1.4
0.50

(a) Complete the table above by calculating enzyme velocity (v) and turnover number (kcat) for each substrate.
(b) Which of these substrates binds most tightly to the enzyme? Explain.
(c) With which substrate does the enzyme exhibit the highest efficiency and specificity? Explain and show your
calculations (if any).
2. The steady state kinetics of the enzyme aspartase were studied in the absence and presence of the inhibitor
hydroxymethylaspartate (A) ([A] = 2.5 x 10-4 M). The initial rate (v) is given as a function of substrate
concentration.
[S]
(M)
1 x 10 -4
5 x 10 -4
1.5 x 10 -3
2.5 x 10 4
5 x 10 -3

v (no inhibitor)
(M/min)
0.026
0.092
0.136
0.150
0.165

v (inhibitor present)
(M/min)
0.010
0.040
0.086
0.120
0.142

(a) Draw a Lineweaver-Burk plot for the enzyme reaction

Use your graph to determine
(b) Km in the absence of inhibitor A
(c) Km in the presence of inhibitor A
(d) Vmax in the absence of inhibitor A
(e) Vmax in the presence of inhibitor A
(f) the type of inhibition
3. From a Lineweaver-Burk plot of an enzyme,
at 1/v = 0.
(a) Show a graphical representation
(b) Calculate Km
(c) Calculate Vmax

1/v = 25.0 hr/mol at 1/[S] = 0

and

1/[S] = - 1.13 x 102 L/mol

4. Another way of expressing enzyme kinetic data is to plot v vs v/[S] which is known as the Eadie-Hofstee plot.
(a) Rearrange the Michaelis - Menten equation to give v as a function of v/[S].
(b) What is the function of the slope? the y-intercept? the x-intercept?
5. What advantage can be gained by synthesizing proteolytic enzymes as zymogens?
6. Identify an enzyme used to diagnose a specific human disorder or disease. Discuss its biochemistry and use in
clinical practice. Cite your references.