Enzymes: Principles of Catalysis
Lect 15 Week 31
COURSEWORK: Set in: Due in: Worth: Size: What:
Week 19 (Fri 6th Dec 2013) Week 32 (Fri 7th March 2014) 50% unit mark A2 poster
I would like you to prepare an A2 poster on any interesting enzyme that you can find in the Protein Data Bank (PDB) or BRENDA. We’ve had a look at the PDB and BRENDA, and these are useful databases to know. I’m looking for a clear, visual presentation, not text heavy. This is also good practice for your 3rd yr posters next year. About 5 references Focus on the structure and function of the enzyme, but don’t try and cover everything on it! Your audience Your audience is a postgrad student at a conference who doesn’t know the enzyme and is interested in its biology. So colourful figures and good legends are important. Try and use CHIMERA to make some nice images for the figures. Marked out of 100 Important aspects are content, design, presentation, flow, use of text, use of graphics. Note: posters usually a few £, but you don’t have to laminate them.
Worked examples Two examples on ES interactions that we will work through – calculators out!
Worked example (i) You obtain the following initial-rate data for two forms of ribonuclease enzyme:
What are you going to plot?
Michaelis-Menten kinetics of wildtype & mutant RNase 100.00
Vmax?
wildtype
90.00 80.00 70.00
V0
60.00 50.00 40.00 30.00
Vmax?
20.00
mutant
10.00 0.00 0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
[S] What are the approx Vmax and Km values for each enzyme? How can we get more precise values?
Vmax? V0
[S] Answer: Lineweaver-Burk (LB) Plot Linearised, Double-Reciprocal
Lineweaver-Burk plot of wildtype and mutant RNase 0.50
mutant
0.45 0.40 0.35
1/V0
0.30 0.25 0.20 0.15
wildtype
0.10 0.05 0.00 0.00
2.00
4.00
6.00
1/[S]
8.00
10.00
12.00
Lineweaver-Burk plot of wildtype and mutant RNase 0.50
mutant
0.45 0.40 0.35
1/V0
0.30 0.25 0.20 0.15
wildtype
0.10 0.05 0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
1/[S] Wildtype:
Mutant:
1/V0 = 0.01
1/V0 = 0.05
-1/[S] = -1.66
-1/[S] = -0.37
Lineweaver-Burk plot of wildtype and mutant RNase 0.50
mutant
0.45 0.40 0.35
1/V0
0.30 0.25 0.20 0.15
wildtype
0.10 0.05 0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
1/[S] Wildtype:
Mutant:
1/V0 = 0.01
1/V0 = 0.05
-1/[S] = -1.66
-1/[S] = -0.37
Calculate Vmax , Km and kcat for each enzyme.. (assume the enzyme was at 1 nM)
Lineweaver-Burk plot of wildtype and mutant RNase
100.00
0.50
90.00
0.45
80.00
0.40
70.00
0.35
60.00
0.30
1/V0
V0
Michaelis-Menten kinetics of wildtype & mutant RNase
50.00 40.00 30.00
0.25 0.20 0.15
20.00
0.10
10.00
0.05
0.00 0.00
5.00
10.00
15.00
20.00
[S]
25.00
30.00
35.00
0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
1/[S]
Vmax = 100 µM/min Km = 0.602 µM
Vmax = 20 µM/min Km = 2.68 µM
Worked example (ii) You obtain the following initial-rate data for the enzyme lambda exonuclease. This enzyme digests DNA:
[DNA] in nM 0.63 1.25 2.50 5.00 10.00 20.00
Initial rate, v0, in nM/min 4.14 6.32 7.81 8.95 9.80 10.24
How will you determine Vmax, Km and kcat?
0.26 0.24
10
0.2
8
0.18
1 / Rate
Rate (nM/min)
0.22
6 4
0.16 0.14 0.12 0.1 0.08 0.06
2
0.04 0.02
0
0
0
2
4
6
8
10 12 14 16 18 20
-1.2
-0.9
-0.6
[DNA] in nM
-0.3
0
0.3
0.6
0.9
1.2
1 / [Substrate] Parameter Vmax Km
Value
Std. Error
10.7126 0.9357
0.0991 0.0377
1.5
1.8
COURSEWORK: Set in: Due in: Worth: Size: What:
Week 19 (Fri 6th Dec 2013) Week 32 (Fri 7th March 2014) 50% unit mark A2 poster
I would like you to prepare an A2 poster on any interesting enzyme that you can find in the Protein Data Bank (PDB) or BRENDA. We’ve had a look at the PDB and BRENDA, and these are useful databases to know. I’m looking for a clear, visual presentation, not text heavy. This is also good practice for your 3rd yr posters next year. About 5 references Focus on the structure and function of the enzyme, but don’t try and cover everything on it! Your audience Your audience is a postgrad student at a conference who doesn’t know the enzyme and is interested in its biology. So colourful figures and good legends are important. Try and use CHIMERA to make some nice images for the figures. Marked out of 100 Important aspects are content, design, presentation, flow, use of text, use of graphics. Note: posters usually a few £, but you don’t have to laminate them.
Worked examples Two examples on ES interactions that we will work through – calculators out!
Worked example (i) You obtain the following initial-rate data for two forms of ribonuclease enzyme:
What are you going to plot?
Michaelis-Menten kinetics of wildtype & mutant RNase 100.00
Vmax?
wildtype
90.00 80.00 70.00
V0
60.00 50.00 40.00 30.00
Vmax?
20.00
mutant
10.00 0.00 0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
[S] What are the approx Vmax and Km values for each enzyme? How can we get more precise values?
Vmax? V0
[S] Answer: Lineweaver-Burk (LB) Plot Linearised, Double-Reciprocal
Lineweaver-Burk plot of wildtype and mutant RNase 0.50
mutant
0.45 0.40 0.35
1/V0
0.30 0.25 0.20 0.15
wildtype
0.10 0.05 0.00 0.00
2.00
4.00
6.00
1/[S]
8.00
10.00
12.00
Lineweaver-Burk plot of wildtype and mutant RNase 0.50
mutant
0.45 0.40 0.35
1/V0
0.30 0.25 0.20 0.15
wildtype
0.10 0.05 0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
1/[S] Wildtype:
Mutant:
1/V0 = 0.01
1/V0 = 0.05
-1/[S] = -1.66
-1/[S] = -0.37
Lineweaver-Burk plot of wildtype and mutant RNase 0.50
mutant
0.45 0.40 0.35
1/V0
0.30 0.25 0.20 0.15
wildtype
0.10 0.05 0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
1/[S] Wildtype:
Mutant:
1/V0 = 0.01
1/V0 = 0.05
-1/[S] = -1.66
-1/[S] = -0.37
Calculate Vmax , Km and kcat for each enzyme.. (assume the enzyme was at 1 nM)
Lineweaver-Burk plot of wildtype and mutant RNase
100.00
0.50
90.00
0.45
80.00
0.40
70.00
0.35
60.00
0.30
1/V0
V0
Michaelis-Menten kinetics of wildtype & mutant RNase
50.00 40.00 30.00
0.25 0.20 0.15
20.00
0.10
10.00
0.05
0.00 0.00
5.00
10.00
15.00
20.00
[S]
25.00
30.00
35.00
0.00 0.00
2.00
4.00
6.00
8.00
10.00
12.00
1/[S]
Vmax = 100 µM/min Km = 0.602 µM
Vmax = 20 µM/min Km = 2.68 µM
Worked example (ii) You obtain the following initial-rate data for the enzyme lambda exonuclease. This enzyme digests DNA:
[DNA] in nM 0.63 1.25 2.50 5.00 10.00 20.00
Initial rate, v0, in nM/min 4.14 6.32 7.81 8.95 9.80 10.24
How will you determine Vmax, Km and kcat?
0.26 0.24
10
0.2
8
0.18
1 / Rate
Rate (nM/min)
0.22
6 4
0.16 0.14 0.12 0.1 0.08 0.06
2
0.04 0.02
0
0
0
2
4
6
8
10 12 14 16 18 20
-1.2
-0.9
-0.6
[DNA] in nM
-0.3
0
0.3
0.6
0.9
1.2
1 / [Substrate] Parameter Vmax Km
Value
Std. Error
10.7126 0.9357
0.0991 0.0377
1.5
1.8
