CHMI 2227 - E.R. Gauthier, Ph.D. 1

CHMI 2227EBiochemistry I

Enzymes:- Kinetics

CHMI 2227 - E.R. Gauthier, Ph.D. 2

Enzymatic reactions

Enzyme (each = 1 µmol)

Substrate (each = 1 µmol)

Only concentrations we know we’re the ones who set up the experiment!

X min Product

CHMI 2227 - E.R. Gauthier, Ph.D. 3

Enzymatic reactionsHow do we measure enzyme activity?

1. Detection of the product(s): pNA = para-nitroaniline Absorbs at 405 nm

H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-OH

O O O O

CH2

COO-

CH2

CH2

COO-

CHCH3H3C

CH2

COO-

NO2H2N

pNA (yellow)

H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NH-

O O O O

CH2

COO-

CH2

CH2

COO-

CHCH3H3C

CH2

COO-

NO2DEVD-pNA(uncolored)

DEVD(uncoloured)

Caspase 3 (proteasehydrolase)Measure increase

in A405nm

CHMI 2227 - E.R. Gauthier, Ph.D. 4

Enzymatic reactionsHow do we measure enzyme activity? 2. Accumulation/utilisation of a co-factor:

NADH = absorbs strongly at 340 nm ( = 6.3 molL-1cm-1 ) NAD+ =does not absorb at 340 nm

Measure increase in A340nm

Measure decrease in A340nm

Lactate dehydrogenase

CHMI 2227 - E.R. Gauthier, Ph.D. 5

Enzymatic reactionsHow do we measure enzyme activity? 3. Coupled reactions:

Very useful when neither substrate/product/co-factor can be (easily) detected;

Glutaminase

+ NH4+

1st reaction

Measure increase in A340nm

GlutamateDehydrogenase

+ NAD+ + NADH +H+

2nd reaction

+ NH4++ H2O

Detectable by HPLC but not practical

CHMI 2227 - E.R. Gauthier, Ph.D. 6

Enzymatic reactions

1 min

3 µmol / min VELOCITYor Rate

15 µmol S vs 1 µmol E

Time

[Pro

duct

]

Slope = Initial velocity = v0 = [P] / time

2 min

3 µmol / min

4 min

<3 µmol / min

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Enzymatic reactions

Time

[Pro

duct

]

v0 is proportional to [E]

1µmol E

2µmol E

3µmol E

1 min

3 µmol / min

15 µmol S vs 1 µmol E

1 min

6 µmol / min

15 µmol S vs 2 µmol E

1 min

9 µmol / min

15 µmol S vs 3 µmol E

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Enzymatic reactions

1 min

2 µmol / min

1 µmol / min

1 min

3 µmol / min

1 min

[Substrate]

v 0

Maximum velocity = Vmax

Vmax

½ Vmax

E saturated by S

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Enzymatic reactions

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Michaelis-Menten Equation

[Substrate]v 0

Maximum velocity = Vmax

Vmax

½ Vmax

vo = Vmax [S] Km + [S]

CHMI 2227 - E.R. Gauthier, Ph.D. 11

Michaelis-Menten Equation

[Substrate]

v 0

Vmax

½ Vmax

Km1 Km2

E2

E1

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Km

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Turnover number

E + S ES E + Pk2

k1

K-1

FAST SLOW

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Measuring Km and Vmax

[Substrate]

v 0

Vmax

½ Vmax

Km

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Measuring Km and Vmax

1/vo

1/[S]

1/Vmax

-1/Km

Lineweaver-Burk plot

1 vo

= Km x 1 + 1

Vmax [S] Vmax