Reaction: 2-phosphoglycerate ↔ phosphoenolpyruvate + H2O
Also an enzyme in glycolysis
Chemistry 340 Exam 2 Lectures 6-7 |
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Enzyme Catalysis
Major concepts:
1. What is a serine protease, and what mechanism(s) does it use to catalyze proteolysis? How can you distinguish one transition state from another, and what happens to (a) produce the first transition state and (b) produce the second transition state?
2. How are the mechanisms used by enolase and carbonic anhydrase similar? How are the enzymes different?
Core knowledge:
1. What is the catalytic triad of a serine protease, and what type of bi-substrate reaction does a serine protease catalyze? What kinetic studies provide evidence for mechanisms? What are the specificity pocket and the oxyanion hole, and how are they involved in the reaction?
2. What is a kinase, and what mechanism(s) does hexokinase use to catalyze its reaction?
3. What reaction is catalyzed by enolase, and what co-factor(s) and active site residues are required?
4. What reaction is catalyzed by carbonic anhydrase, and what co-factor(s) and active site residues are required?
General characteristics, reviewed:
A. Enzymes stabilize the transition state by specific interactions between the transition state
and the active site.
B. The mechanisms of enzyme catalysis are acid-base, covalent, and metal ion.
C. Bi-substrate reactions may involve a ternary complex or be Ping-Pong.
Examples of enzyme catalysis require
A. Kinetic studies with pure enzyme, optimal substrates, modified substrates, inhibitors
B. Enzyme primary and tertiary or quaternary structure
C. Locating the active site and the residues involved in catalysis
D. Visualization of the active site and the way it changes:
1. No substrate bound = empty active site
2. Substrate binds
3. Transition state(s) forms
4. Product forms
5. Product released = ready to repeat
Chymotrypsin is a digestive enzyme,
A. synthesized by the pancreas (inactive), active in the small intestine
B. a protease that adds H2O to break a peptide bond
C. specificity: breaks bonds on the carboxyl side of Phe or Tyr
Structure: 3 protein chains with several disulfide bonds
Active site = cleft with Ser, His, and Asp side chains (catalytic triad)
Kinetic studies
A. Using an artificial substrate = S1 → P1 + acetate (P2)
1. Experimental observations:
Initial burst (high slope) followed by a slower rate (lower slope),
plotting time vs mole P1/mole enzyme
2. Analysis: The reaction for chymotrypsin is
E–OH + S1 ↔ E–acetate + P1 → E–acetate + S2 (H2O) → E–OH + acetate
B. Effect of pH on rate
1. Experimental observations:
a. maximum rate at pH 8 using low [S] – Measures kcat/KM because [S] is low.
b. Maximum rate at high [S] gives kcat, and plotting 1/kcat → low rate below pH 7.
c. Determining KM at each pH and plotting 1/KM → low rate above pH 9.
2. Analysis: At low pH His57 side chain is protonated and can't participate in the reaction.
At high pH Ile 16–NH3+ → NH2, resulting in loss of a salt bridge to Asp- and
conformation change → substrate can no longer bind properly.
3. Normally Ser-OH would not lose H+, but His57 proximity to Asp102 makes it attract H+
from Ser195 .
Process of the reaction: Mechanism Figure 6-21
1. Catalytic triad = Asp102 – His57 – Ser195 connected by hydrogen bonds in the active site.
2. Substrate binds, with aromatic side chain in the hydrophobic pocket (specificity pocket)
positioning the peptide bond with C=O next to Ser195 O–H and –NH– next to His57.
3. His57 attraction of Ser195O– H → Ser195 O- which then attacks the substrate C=O →
4. Oxyanion intermediate (short-lived), with C–O- stabilized by two N–H's →
5. –NH– attracts His57 – H and forms H – NH – product 1, which is released.
Ser195 O has a covalent bond to the substrate –O–C–, which remains in the active site.
= acyl–enzyme intermediate
6. H –OH (S2) enters the active site oriented so that H – is near His57 & O is near C=O →
7. Oxyanion intermediate, again with C–O- stabilized by two N–H's →
8. Product 2 – CO O- with Ser195–O–H regenerated.
Chymotrypsin Mechanisms used = acid-base and covalent catalysis
No co-factor required.
Bi-substrate reaction type = Ping-Pong
Chymotrypsin is a hydrolase.
Hexokinase (a transferase)
Reaction: glucose + ATP4- → glucose-6-phosphate + ADP3-
ATP4- and ADP3- have Mg2+ associated with the negatively charged phosphates
First enzyme in glycolysis pathway
More favorable reaction: ATP + H2O → ADP + Pi (inorganic phosphate)
Transfer of phosphate to glucose requires excluding H2O from the active site.
Process of the reaction:
1. Glucose and ATP . Mg bind in the active site.
2. Enzyme changes conformation (induced fit) and closes, excluding H2O. Fig. 6-22
3. Enzyme catalyzes transfer of phosphate from ATP to glucose carbon 6.
Significant aspects of the hexokinase reaction:
Mg2+ is required, associated with ATP. Functions to shield ATP's negative charges from
glucose carbon 6-OH.
Converting ATP to ADP is favorable = thermodynamically spontaneous, but
hexokinase (active conformation) is required for a noticeable reaction rate.
Bi-substrate reaction that involves a ternary complex.
Sugar (glucose or xylose) binding triggers the hexokinase conformation change →
transfer of phosphate from ATP.
Enolase
Reaction: 2-phosphoglycerate ↔ phosphoenolpyruvate + H2O
Also an enzyme in glycolysis
Active site
1. Requires 2 Mg2+ to attract COO- of 2-phosphoglycerate
2. Lys side chain–NH3+ next to Glu side chain–COO- ⇔ Lys–NH2: and Glu–COOH
3. Lys–NH2: acts as a base to remove 2-phosphoglycerate H+ to start the reaction.
4. Glu–COOH provides H+ to make it easier to remove OH- from 2-phosphoglycerate.
5. Regenerating the enzyme requires transferring H+ from Lys to Glu.
Significant aspects of the enolase reaction:
Mg2+ is required as a co-factor. No coenzyme required.
Metal ion and acid-base catalysis
pKa's for both Lys and Glu are modified by their proximity in the active site.
1 substrate and 2 products
Enolase is a lyase.
Carbonic anhydrase
Reaction: H2O + CO2 ⇔ HCO3- + H+
Found in red blood cells, plasma; important for CO2 transport by blood (kcat ∼ 106 s-1 at 25°C)
