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CHMI 2227E Biochemistry I

CHMI 2227E Biochemistry I. Enzymes: Kinetics. X min. Product. Enzymatic reactions. Let’s set up a typical enzymatic reaction:. 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

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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 • Let’s set up a typical enzymatic reaction: 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 • So: • 1) v0 (initial velocity) is the rate of the reaction very early on  when [P] is negligeable; • 2) v0 can be obtained by taking the slope of the graph of [P] vs Time (units: concentration / time) • 3) v0 varies as a function of [E]; • 4) v0 increases as a function of [S] UNTIL E is saturated by S. • 5) When E is saturated with S  v0 = Vmax CHMI 2227 - E.R. Gauthier, Ph.D.

  10. The relationship between vo and [S] can be viewed as a 2 step reaction: This relationship can be expressed by the Michaelis-Menten equation: Maximum velocity = Vmax Vmax k1 k2 k-1 ½ Vmax FAST SLOW v0 [Substrate] vo = Vmax [S] Km + [S] E + S ES E + P Michaelis-Menten Equation CHMI 2227 - E.R. Gauthier, Ph.D.

  11. E1 Vmax E2 ½ Vmax v0 [Substrate] Km2 Km1 Michaelis-Menten Equation • Km can be calculated as the [S] required to acheive half the Vmax; • Km is a measure of the affinity of E for S: • The lower the Km, the less S is requried by E to acheive ½ Vmax, and the greater the affinity of E for S. 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 • At saturating [S] : • vo = Vmax • vo is determined by [E] • k2 will drive the rate; • k2= kcat • So: Vmax = kcat [E]total • kcat = Vmax/[E]total • kcat = turnover number = maximum number of substrate molecules converted to product per second by each active site (units = s-1) • 1/kcat = amount of time required for E to convert 1 substrate molecule to the product (i.e. time for 1 catalytic event). Units: s. CHMI 2227 - E.R. Gauthier, Ph.D.

  14. Neither Km nor Vmax can be easily obtained directly from kinetic data because Vmax is rarely acheived (its an hyperbolic curve…); 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 • However, Km and Vmax can be easily obtained if we take the reciprocal of (and slightly rearrange) the Michaelis-Menten equation: the Lineweaver-Burk equation: • The graph of 1/vo vs 1/[S] gives a straight line with: • Intercept on the x axis = -1/Km • Intercept on the y axis = 1/Vmax • This is the BEST and EASIEST way to accurately obtain Vmax and Km since: • You know [S] (you’re the one who did the experiment!!) • V0 is easily obtained in the lab (slope of [P] vs Time). CHMI 2227 - E.R. Gauthier, Ph.D.

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