Enzyme Kinetics: Km

BIOLOGY 371

INDEPENDENT RESEARCH

WEEK 7

Determination of Km - Coenzyme

1. Use seven concentrations. * Useful dilutions are 2, 4, 8, 12, 16 and 20.
2. When preparing assays add 10 µl of NADP to assay buffer, then add enzyme solution [NADP⁺ stabilizes enzyme during incubation at 30° C].
3. Initiate reaction by adding substrate.

*Example of Concentrated Solutions:

If the assay calls for 0.18 mM NADP⁺:

1 M NADP⁺ = 765.4 g / litter
1 mM NADP⁺ = 765.4 mg / liter = 0.7654 mg / mL
0.18 mM NADP⁺ = 0.1378 mg / mL

Our procedure will add 10 ul of concentrated NADP⁺ solution to 1 mL of assay buffer. These 10 ul must contain 0.1378 ug of NADP⁺ and thus our concentrated NADP⁺ solution must have 13.78 ug / mL.
Thus 10 µl of 13.78 µg / mL in 1 mL assay = 0.18 mM

Determining Km for NADP+

1. Use seven concentrations of substrate. Dilute original concentration* by 2, 4, 8, 12, 16, 20.
2. Prepare enough assay buffer to run all concentrations in triplicate. Place in water bath.
3. Run triplicate assays for all concentrations, according to standard assay procedures. Be careful, rinse cuvettes well, and change pipet tips between
each assay.
4. Repeat the whole procedure two times, i.e., the final Km will be the mean of three determinations ± S. E. of the mean [ see Appendix].

*Prepare substrates in concentrated form using 1.5 mL centrifuge tubes.
Since an assay requires 10 ul of substrate solution per mL of assay solution, these concentrated solutions must be 100 X's the assay concentration.

Calculation of Km

1. Determine OD / min for each assay.
2. Determine mean OD / min for each concentration of substrate or co-enzyme [slope @ 1A].
3. Determine Vo for each concentration. Vo = OD / Min [0.1613]
4. Lineweaver-Burke Plot: Plot 1 / Vo vs [S]
5. Hofstee Plot: Plot Vo vs Vo / [S]

Table 2. Kinetics of Substrate Utilization for Cauliflower IDH

Substrate Km +/- S.E. of the Mean Number of Determinations

Isocitrate 5.34 +/- 0.49 X 10-5 M 4

NADP+ 10.5 +/- 0.29 X 10-6 M 4

Data of Tom Beadle `98, Byron Faler `98 and Sean Keesee `98

WORKSHEET

Dilutions of NADP+ for Km

Tube Dilution ul [1] ul H2O Molarity 1 / [S] Vo 1 / Vo

1 0 100 0

2 4 25 75

3 8 20 140

4 12 10 110

5 16 10 150

6 20 10 190

7 24 10 230

Return to Schedule

Substrate	Km +/- S.E. of the Mean	Number of Determinations
Isocitrate	5.34 +/- 0.49 X 10-5 M	4
NADP+	10.5 +/- 0.29 X 10-6 M	4

Tube	Dilution	ul [1]	ul H2O	Molarity	1 / [S]	Vo	1 / Vo
1	0	100	0
2	4	25	75
3	8	20	140
4	12	10	110
5	16	10	150
6	20	10	190
7	24	10	230