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 Enzyme Nonenzymatic reaction rate (s -1 ) Enzymatic reaction rate (s -1 ) Rate enhancement Carbonic anhydrase 1.3 x x x 10 6 Triose phosphate isomerase 4.3 x x 10 9 Staphlococcal nuclease 1.7 x x 10 14

Enzymes Enzyme Action Figure 1-1 Substrate - acted on by enzyme, high specificity Enzyme - called ____-ase (protease), ES complex very specific, small amt needed, remade

Enzymes Substrate specificity Types of complementarity between enzyme and substrate: Geometric Electronic Hydrophobic Hydrophilic Substrate binding sites undergo conformational change when substrate binds induced fit “lock-and-key”

Enzymes E + SESE + P  G’˚ < 0; favorable

Enzymes Enzymes affect reaction rates, not equilibria Catalysts enhance reaction rates by lowering activation energy Rate is set by activation energy  G ‡ Higher activation energy --> slower reaction Overall rate of reaction is determined by step with highest activation energy --> rate-limiting step

Enzymes Measure rate of catalytic reaction Velocity of rxn measured by decrease in [substrate] over time OR increase in [product] over time Graph linear at first, then flattens out and no more product is made Can determine velocity of reaction using early data where graph is linear Initial Rate (V 0 ) = slope of line Determine rate

Enzymes Influences on rate of reaction 1. Amount of enzyme Initial velocity (V 0 )  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 V 0 = 10 nmol of prod / min THEN a cell extract that yields 20 nmol of prod / min has 2 µg

Enzymes Influences on rate of reaction 2. Temperature Enzymes are proteins so they will be denatured at T > ˚C most 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 ( ˚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.

Enzymes Influences on rate of reaction 3. pH Activity 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

Enzymes Influences on rate of reaction 4. [substrate] Low [substrate] - active site not sat’d, so enzyme NOT working at full capacity High [substrate] - no open sites, enzyme working at full capacity (Vmax)

Enzymes Influences 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 + SESE + P k1k1 k -1 k2k2 k -2 Slow step Rate-limiting

Enzymes For experimental determination of Km and Vmax do Lineweaver-Burk plot plot 1/V 0 vs. 1/[S] Straight line --> slope, y-intercept, x-intercept More accurate determination of V max

Enzymes OUR 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 Products NITROPHENOL PHOSPHATE  NITROPHENOL + PHOSPHATE (colorless in alkaline)(yellow in alkaline)

Enzymes OUR ENZYME - WHEAT GERM ACID PHOSPHATASE Exp 1 Make wheat germ extract Reaction time course with pure AP and wheat germ AP Nitrophenol standards to create std. curve Determine V 0 of rxn catalyzed by pure AP and wheat germ AP Estimate amount of AP enzyme in wheat germ Exp 2Look at effects of [substrate] on velocity of rxns Reaction time course with pure AP and varying amounts of substrate Plot MM and LB curves Calculate Vmax and Km