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CHMI 2227E Biochemistry I

CHMI 2227E Biochemistry I. Enzymes: Kinetics. X min. Product. Enzymatic reactions. Enzyme (each = 1 µmol). Only concentrations we know  we’re the ones who set up the experiment!. Substrate (each = 1 µmol). O. O. O. O. DEVD-pNA (uncolored). H 3 + N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NH-.

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CHMI 2227E Biochemistry I

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

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

  3. O O O O DEVD-pNA (uncolored) H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-NH- NO2 CH2 COO- CH2 CH2 COO- CH CH2 COO- H3C CH3 Measure increase in A405nm Caspase 3 (proteasehydrolase) O O O O DEVD (uncoloured) H3+N-CH-C-NH-CH-C-NH-CH-C-NH-CH-C-OH NO2 H2N CH2 COO- CH2 CH2 COO- CH CH2 COO- H3C CH3 pNA (yellow) Enzymatic reactionsHow do we measure enzyme activity? • 1. Detection of the product(s): • pNA = para-nitroaniline  Absorbs at 405 nm CHMI 2227 - E.R. Gauthier, Ph.D.

  4. Lactate dehydrogenase Measure decrease in A340nm Enzymatic reactionsHow 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 CHMI 2227 - E.R. Gauthier, Ph.D.

  5. Detectable by HPLC but not practical Glutaminase 1st reaction + NH4+ 2nd reaction Glutamate Dehydrogenase Measure increase in A340nm + NADH +H+ + NAD+ + H2O + NH4+ Enzymatic reactionsHow do we measure enzyme activity? • 3. Coupled reactions: • Very useful when neither substrate/product/co-factor can be (easily) detected; CHMI 2227 - E.R. Gauthier, Ph.D.

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

  7. 3 µmol / min 1 min v0 is proportional to [E] 15 µmol S vs 1 µmol E 3µmol E 2µmol E 6 µmol / min [Product] 1 min 1µmol E 15 µmol S vs 2 µmol E Time 9 µmol / min 1 min 15 µmol S vs 3 µmol E Enzymatic reactions CHMI 2227 - E.R. Gauthier, Ph.D.

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

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

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

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

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

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

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

  15. 1/vo 1/Vmax 1/[S] = Km x 1 + 1 1 vo [S] Vmax -1/Km Vmax Lineweaver-Burk plot Measuring Km and Vmax CHMI 2227 - E.R. Gauthier, Ph.D.

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