1 / 15

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-.

nariko
Télécharger la présentation

CHMI 2227E Biochemistry I

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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.

More Related