Data Table for Enzyme Dilutions (Table 1)

Trial

Cocktail A
1
2
3
4
5
6
7

Tris 0.2mL
NADH 0.3mL
H20 1.9mL

Pyruvate (ml) [pyruvate] (mM) water (ml) enzyme (ml)

0.025
0.05
0.475
0.1
0.050
0.10
0.450
0.1
0.100
0.20
0.400
0.1
0.200
0.40
0.300
0.1
0.050
1.00
0.450
0.1
0.100
2.00
0.400
0.1
0.250
5.00
0.250
0.1

Data Table for Enzyme Dilutions (Table 1) - TF Data

Trial
Cocktail A
Pyruvate (ml) [pyruvate] (mM) water (ml) enzyme (ml)
1
0.025
0.05
0.475
0.1
2
0.050
0.10
0.450
0.1
3
Tris 0.2mL
0.100
0.20
0.400
0.1
4 NADH 0.3mL
0.200
0.40
0.300
0.1
5
H20 1.9mL
0.050
1.00
0.450
0.1
6
0.100
2.00
0.400
0.1
7
0.250
5.00
0.250
0.1

dilution factor A340/min

100
0.015
100
0.021
100
0.031
100
0.044
100
0.040
100
0.037
100
0.045

dilution factor A340/min

1000
0.054
1000
0.085
1000
0.201
1000
0.308
1000
0.508
1000
0.614
1000
0.692

Activity versus Substrate

Trial

Cocktail A

Pyruvate (ml) [pyruvate] (mM) water (ml)

1
0.025
0.05
0.475
2
0.050
0.10
0.450
3 Tris 0.2mL
0.100
0.20
0.400
4 NADH 0.3mL
0.200
0.40
0.300
5 H20 1.9mL
0.050
1.00
0.450
6
0.100
2.00
0.400
7
0.250
5.00
0.250

enzyme (ml)
0.1
0.1
0.1
0.1
0.1
0.1
0.1

Activity Versus Substrate (Table 2) - TF Data

Trial
Cocktail A Pyruvate (ml) [pyruvate] (mM) water (ml)
1
0.025
0.05
0.475
2
0.050
0.10
0.450
3 Tris 0.2mL
0.100
0.20
0.400
4 NADH 0.3mL
0.200
0.40
0.300
5 H20 1.9mL
0.050
1.00
0.450
6
0.100
2.00
0.400
7
0.250
5.00
0.250

enzyme (ml)
0.1
0.1
0.1
0.1
0.1
0.1
0.1

dilution factor
100
100
100
100
100
100
100

A340/min
0.015
0.021
0.031
0.044
0.040
0.037
0.045

dilution factor
1000
1000
1000
1000
1000
1000
1000

A340/min
0.054
0.085
0.201
0.308
0.508
0.614
0.692

Data Table for Inhibition Studies (Table 3)

Sample
Cocktail B
Pyruvate (mL) [pyruvate] (mM) water (mL) enzyme (mL)
1
0.025
0.05
0.475
0.1
2
Tris 0.2mL
0.050
0.10
0.450
0.1
3 NADH 0.3mL
0.100
0.20
0.400
0.1
4
H20 1.7mL
0.200
0.40
0.300
0.1
5 Inhibitor B 0.2mL
0.050
1.00
0.450
0.1
6
0.100
2.00
0.400
0.1
7
0.250
5.00
0.250
0.1

Data Table for Inhibition Studies (Table 3) - TF Data

Sample
Cocktail B
Pyruvate (mL) [pyruvate] (mM) water (mL) enzyme (mL)
1
0.025
0.05
0.475
0.1
2
Tris 0.2mL
0.050
0.10
0.450
0.1
3 NADH 0.3mL
0.100
0.20
0.400
0.1
4
H20 1.7mL
0.200
0.40
0.300
0.1
5 Inhibitor B 0.2mL
0.050
1.00
0.450
0.1
6
0.100
2.00
0.400
0.1
7
0.250
5.00
0.250
0.1

dilution factor A340/min

100
0.000
100
0.000
100
0.009
100
0.000
100
0.000
100
0.000
100
0.003

dilution factor A340/min

100
0.055
100
0.097
100
0.169
100
0.278
100
0.542
100
0.507
100
0.539

1/V0 and 1/[S]

Without Inhibitor

Trial #
1
2
3
4
5
6
7

Substrate
Concentration
(mM)
0.025
0.050
0.100
0.200
0.050
0.100
0.250

A340/min
diluted
0.015
0.021
0.031
0.044
0.040
0.037
0.045

A340/min
undiluted
1.50
2.10
3.10
4.40
4.00
3.70
4.50

V0 without
inhibitor
0.214
0.300
0.442
0.628
0.571
0.528
0.642

V0 with
inhibitor
0.031
0.043
0.063
0.090
0.081
0.075
0.092

Rate in mM
0.214
0.300
0.442
0.628
0.571
0.528
0.642

With Inhibitor
A340/min
diluted
0.000
0.000
0.009
0.000
0.000
0.000
0.003

A340/min
undiluted
0.000
0.000
0.900
0.000
0.000
0.000
0.300

With
[S]
0.05
0.10
0.20
0.40
1.00
2.00
5.00

TF Data
Without Inhibitor

Trial #
1
2
3
4

Substrate
Concentration
(mM)
0.025
0.050
0.100
0.200

A340/min
diluted
0.054
0.085
0.201
0.308

A340/min
undiluted
54
85
201
308

Rate in mM
8.710
13.710
32.419
49.677

With Inhibitor
A340/min
diluted
0.055
0.097
0.169
0.278

A340/min
undiluted
55
97
169
278

5
6
7

0.050
0.100
0.250

0.508
0.614
0.692

508
614
692

V0 without
inhibitor
8.710
13.710
32.419
49.677
81.935
99.032
111.613

V0 with
inhibitor
8.871
15.645
27.258
44.839
87.419
81.774
86.935

TF Data Plot
[S]
0.05
0.10
0.20
0.40
1.00
2.00
5.00

81.935
99.032
111.613

0.542
0.507
0.539

542
507
539

Michaelis-Menten Plot With and Witho

ith Inhibitor
0.800

Rate in mM
0.000
0.000
0.128
0.000
0.000
0.000
0.043

0.700

y = 0.0854ln(x) + 0.5421
R = 0.7351

0.600

V0 in mM/min

0.500

0.400

0.300

0.200
y = 0.0122ln(x) + 0.0773
R = 0.7351
0.100

0.000
0.00

1.00

2.00

3.00

4.00

[S] in mM

ith Inhibitor

Michaelis-Menten Plot With and Without Inhibitor (TF

Rate in mM
8.871
15.645
27.258
44.839

140.000

120.000

y = 24.732ln(x) + 76.236
R = 0.9815

87.419
81.774
86.935
V0 in mM/min

100.000

y = 19.93ln(x) + 66.112
R = 0.9073

80.000

60.000

40.000

20.000

0.000
0.00

1.00

2.00

3.00
[S] in mM

4.00

5.00

Plot With and Without Inhibitor

0854ln(x) + 0.5421

V0 without inhibitor
V0 with inhibitor
Log. (V0 without inhibitor)
Log. (V0 with inhibitor)

y = 0.0122ln(x) + 0.0773
R = 0.7351

4.00

5.00

ithout Inhibitor (TF Data)

6.00

V0 without inhibitor
V0 with inhibitor
Log. (V0 without inhibitor)
Log. (V0 with inhibitor)

6.00

V0
0.214
0.300
0.442
0.628
0.571
0.528
0.642

V0 with
0.031
0.043
0.063
0.090
0.081
0.075
0.092

1/[S]
20.0
10.0
5.0
2.5
1.0
0.5
0.2

1/V0 without
4.67
3.34
2.26
1.59
1.75
1.89
1.56

1/V0 with
32.8
23.4
15.9
11.2
12.3
13.3
10.9

Lineweaver-Burk Plot With an

35.00

30.00

25.00
1/V0 in min/mM

[S]
0.05
0.10
0.20
0.40
1.00
2.00
5.00

20.00

15.00

10.00

5.00

0.00
-10.0

-5.0

0.0

5.0
1/[S] in 1/mM

V0 without
8.710
13.710
32.419
49.677
81.935
99.032
111.613

1/[S]
1/V0 without
20.0
0.115
10.0
0.073
5.0
0.031

V0 with
8.871
15.645
27.258
44.839
87.419
81.774
86.935
1/V0 with
0.113
0.064
0.037

Lineweaver-Burk Plot With and W

0.140

0.120

0.100
1/V0 in min/mM

TF Data Plot
[S]
0.05
0.10
0.20
0.40
1.00
2.00
5.00

0.080

0.060

0.020
0.012
0.010
0.009

0.022
0.011
0.012
0.012

1/V0 in min/mM

2.5
1.0
0.5
0.2

0.060

0.040

0.020

0.000
-5.0

0.0

5.0

10.0
1/[S] in 1/mM

Burk Plot With and Without Inhibitor

y = 1.11x + 10.884
R = 0.9694

1/V0 without
1/V0 with
Linear (1/V0 without)
Linear (1/V0 with)

y = 0.1583x + 1.5524
R = 0.9694

10.0

15.0

20.0

25.0

1/[S] in 1/mM

urk Plot With and Without Inhibitor (TF DATA)

y = 0.0056x + 0.0075
R = 0.9863
y = 0.0052x + 0.0094
R = 0.9978

1/V0 without
1/V0 with

1/V0 with
Linear (1/V0 without)
Linear (1/V0 without)

10.0
1/[S] in 1/mM

15.0

20.0

25.0

Table 4.) Tabulate initial rate (V0) and concentration of pyruvate [S]

[S]
0.05
0.10
0.20
0.40
1.00
2.00
5.00

V0 without
inhibitor
0.214
0.300
0.442
0.628
0.571
0.528
0.642

V0 with
inhibitor
0.031
0.043
0.063
0.090
0.081
0.075
0.092

1/[S]
20.0
10.0
5.0
2.5
1.0
0.5
0.2

1/V0 without
1/V0
inhibitor
withinhibitor
4.67
32.8
3.34
23.4
2.26
15.9
1.59
11.2
1.75
12.3
1.89
13.3
1.56
10.9

V0 without
inhibitor
8.710
13.710
32.419
49.677
81.935
99.032
111.613

V0 with
inhibitor
8.871
15.645
27.258
44.839
87.419
81.774
86.935

1/[S]
20.0
10.0
5.0
2.5
1.0
0.5
0.2

1/V0 without
inhibitor
0.115
0.073
0.031
0.020
0.012
0.010
0.009

TF Data Plot
[S]
0.05
0.10
0.20
0.40
1.00
2.00
5.00

1/V0 with
inhibitor
0.113
0.064
0.037
0.022
0.011
0.012
0.012

Without Inhibitor

Trial #
1
2
3
4
5
6
7

Substrate
Concentration A340/min
(mM)
diluted
0.025
0.015
0.050
0.021
0.100
0.031
0.200
0.044
0.050
0.040
0.100
0.037
0.250
0.045

A340/min
undiluted Rate in mM
1.50
0.214
2.10
0.300
3.10
0.442
4.40
0.628
4.00
0.571
3.70
0.528
4.50
0.642

With Inhibitor
A340/min
diluted
0.000
0.000
0.009
0.000
0.000
0.000
0.003

A340/min
undiluted
0.000
0.000
0.900
0.000
0.000
0.000
0.300

Rate in mM
0.000
0.000
0.128
0.000
0.000
0.000
0.043

TF Data
Without Inhibitor

Trial #
1
2
3
4
5
6
7

Substrate
Concentration A340/min
(mM)
diluted
0.025
0.054
0.050
0.085
0.100
0.201
0.200
0.308
0.050
0.508
0.100
0.614
0.250
0.692

A340/min
undiluted Rate in mM
54
8.710
85
13.710
201
32.419
308
49.677
508
81.935
614
99.032
692
111.613

With Inhibitor
A340/min
diluted
0.055
0.097
0.169
0.278
0.542
0.507
0.539

A340/min
undiluted
55
97
169
278
542
507
539

Rate in mM
8.871
15.645
27.258
44.839
87.419
81.774
86.935