Enzymes

Most biological catalysts are proteins, (some REALLY COOL ONES are folded RNAs)

Catalysts - change rate of reaction without net change of catalyst

Cells make lots of enzymes

Enzymes SPEED up reactions

EnzymeNonenzymatic reaction rate (s-1)

Enzymatic reaction rate (s-1) Rate enhancement

Carbonic anhydrase

1.3 x 10-1 1 x 106 7.7 x 106

Triose phosphate isomerase

4.3 x 10-6 4300 1 x 109

Staphlococcal nuclease

1.7 x 10-13 95 5.6 x 1014

Enzymes

Enzyme Action

Figure 1-1

Substrate - acted on by enzyme, high specificityEnzyme - called ____-ase (protease), ES complex very specific,

small amt needed, remade

EnzymesSubstrate specificity

Types of complementarity between enzyme and substrate:GeometricElectronicHydrophobicHydrophilic

Substrate binding sites undergo conformational change when substrate binds

induced fit“lock-and-key”

Enzymes

E + S ES E + P

G’˚ < 0; favorable

EnzymesEnzymes affect reaction rates, not equilibriaCatalysts enhance reaction rates by lowering activation energy

Rate is set by activation energy G‡

Higher activation energy --> slower reactionOverall rate of reaction is determined by step with highest activation energy --> rate-limiting step

EnzymesMeasure rate of catalytic reaction

Velocity of rxn measured by decrease in [substrate] over time ORincrease in [product] over time

Graph linear at first, then flattens out and no more product is madeCan determine velocity of reaction using early data where graph is linearInitial Rate (V0) = slope of line Determine rate

EnzymesInfluences on rate of reaction

1. Amount of enzymeInitial velocity (V0) amount of active enzyme

Figure 1-3

From this data can determine amount of enzyme in unknown sample

Example - if 1 µg of pure enzyme has a V0 = 10 nmol of prod / minTHEN a cell extract that yields 20 nmol of prod / min has 2 µg

EnzymesInfluences on rate of reaction

2. Temperature

Enzymes are proteins so they will be denatured at T > 50 - 70 ˚Cmost enzymes - increasing the temp up to 45 ˚C usually causes increase in rate

Enzymes have an “optimum temperature range” where their activity is greatest leading to maximum rate (20 - 40 ˚C)

Most enzymes have an optimum temperature.

A general rule of thumb - a reaction's rate approximately doubles with a 10°C increase in reaction or assay temperature. However, since enzymes are held together by weak non-covalent bonds, at higher temperatures, the enzyme catalyzed rate slows down rather than increases.

EnzymesInfluences on rate of reaction

3. pHActivity influenced by pH - why?Very high or very low pH cause denaturation/inactivity

Most enzymes operate best at neutral pH

BUT today our enzyme - acid phosphatase - has a pH optimum at 4.5

Calculate pH optimum by assaying the enzyme activity in buffers of different pH

Plot of enzyme activity Vs. pH is often "bell shaped" since two different amino acid groups of the enzyme are being titrated to different states of ionization at the different pH values

One of the two possible ionization states of the amino acid side chain is effective in enzyme catalysis

EnzymesInfluences on rate of reaction

4. [substrate]

Low [substrate] - active site not sat’d, so enzyme NOT working at full capacityHigh [substrate] - no open sites, enzyme working at full capacity (Vmax)

EnzymesInfluences on rate of reaction

4. [substrate]

Vmax achieved when saturate enzyme with substrate

Under conditions of defined Temp, pH, ionic strength Km = Kd(if the rate of break down of ES to P is slower than the back reaction of ES dissociating to E + S (in other words, Km = Kd, if k-1 >> k2))

Kd = dissociation constant, [substrate] that yields 1/2 saturation of enz

Km and Kd - affinity

E + S ES E + Pk1

k-1

k2

k-2

Slow stepRate-limiting

EnzymesFor experimental determination of Km and Vmax do Lineweaver-Burk plot

plot 1/V0 vs. 1/[S]

Straight line --> slope, y-intercept, x-interceptMore accurate determination of Vmax

EnzymesOUR ENZYME - WHEAT GERM ACID PHOSPHATASE

Lyse open cells to get wheat germ AP - add enzyme extraction buffer which has detergent NP-40 (what happens here?)

Enzyme extract

AP catalyzes removal of phosphate from macromolecule

Use synthetic substrate, nitrophenol phosphate

Substrate AP ProductsNITROPHENOL PHOSPHATE NITROPHENOL + PHOSPHATE (colorless in alkaline) (yellow in alkaline)

EnzymesOUR ENZYME - WHEAT GERM ACID PHOSPHATASE

Exp 1 Make wheat germ extractReaction time course with pure AP and wheat germ APNitrophenol standards to create std. curveDetermine V0 of rxn catalyzed by pure AP and wheat germ APEstimate amount of AP enzyme in wheat germ

Exp 2 Look at effects of [substrate] on velocity of rxnsReaction time course with pure AP and varying amounts of substratePlot MM and LB curvesCalculate Vmax and Km