CHMI 2227E Biochemistry I Enzymes: Kinetics

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Presentation transcript:

CHMI 2227E Biochemistry I Enzymes: Kinetics CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions Enzyme (each = 1 µmol) Only concentrations we know  we’re the ones who set up the experiment! Substrate (each = 1 µmol) X min Product CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions How 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 CH2 COO- CH CH3 H3C NO2 H2N pNA (yellow) H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NH- DEVD-pNA (uncolored) DEVD (uncoloured) Caspase 3 (proteasehydrolase) Measure increase in A405nm CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions How do we measure enzyme activity? 2. Accumulation/utilisation of a co-factor: NADH = absorbs strongly at 340 nm (e = 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.

Enzymatic reactions How 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 Glutamate Dehydrogenase + NAD+ + NADH +H+ 2nd reaction + H2O Detectable by HPLC but not practical CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions [Product] Time VELOCITY 3 µmol / min or Rate 1 min 15 µmol S vs 1 µmol E Time [Product] Slope = Initial velocity = v0 = [P] / time 2 min 3 µmol / min 4 min <3 µmol / min CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions v0 is proportional to [E] 3µmol E 2µmol E 1 min 3 µmol / min 15 µmol S vs 1 µmol E 6 µmol / min 15 µmol S vs 2 µmol E 9 µmol / min 15 µmol S vs 3 µmol E Time [Product] v0 is proportional to [E] 1µmol E 2µmol E 3µmol E CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions Maximum velocity = Vmax Vmax ½ Vmax v0 [Substrate] 1 µmol / min 1 min [Substrate] v0 Maximum velocity = Vmax Vmax ½ Vmax 2 µmol / min 1 min 3 µmol / min 1 min E saturated by S CHMI 2227 - E.R. Gauthier, Ph.D.

Enzymatic reactions CHMI 2227 - E.R. Gauthier, Ph.D.

Michaelis-Menten Equation [Substrate] v0 Maximum velocity = Vmax Vmax ½ Vmax vo = Vmax [S] Km + [S] CHMI 2227 - E.R. Gauthier, Ph.D.

Michaelis-Menten Equation [Substrate] v0 Vmax ½ Vmax Km1 Km2 E2 E1 CHMI 2227 - E.R. Gauthier, Ph.D.

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

Turnover number E + S ES E + P FAST SLOW k1 k2 K-1 CHMI 2227 - E.R. Gauthier, Ph.D.

Measuring Km and Vmax Vmax ½ Vmax v0 Km [Substrate] CHMI 2227 - E.R. Gauthier, Ph.D.

Measuring Km and Vmax 1/vo 1 = Km x 1 + 1 vo Vmax [S] 1/Vmax 1/[S] Lineweaver-Burk plot 1 vo = Km x 1 + 1 Vmax [S] CHMI 2227 - E.R. Gauthier, Ph.D.