Chapt. 8 Enzymes as catalysts
Ch. 8 Enzymes as catalysts
Student Learning Outcomes:
• Explain general features of enzymes
as catalysts: Substrate -> Product
• Describe nature of catalytic sites
•
general mechanisms
• Describe how enzymes lower
activation energy of reaction
• Explain how drugs and toxins inhibit
enzymes
• Describe 6 categories of enzymes
Catalytic power of enzymes
Enzymes do not invent new
reactions
Enzymes do not change
possibility of reaction to occur
(energetics)
Enzymes increase the rate of
reaction by factor of 1011 or
higher
Fig. 8.1 box of golfballs,
effect of browning enzyme
Enzymes catalyze reactions
• Enzymes provide speed, specificity and regulatory
control to reactions
• Enzymes are highly specific for biochemical
reaction catalyzed (and often particular substrate)
• Enzymes are usually proteins
• (also some RNAs = ribozymes)
• E + S ↔ ES
• ES ↔ EP
binding substrate
substrate converted to bound product
• EP ↔ E + P release of product
Glucokinase is a typical enzyme
Glucokinase is typical enzyme:
• ATP: D-glucose 6-phosphotransferase
• Very specific for glucose
• Not phosphorylate other hexoses
• Only uses ATP, not other NTP
• 3D shape of enzyme critical for its
function (derived from aa sequence)
Fig. 8.2 glucokinase
A. Active site of enzyme
• Enzyme active site does catalysis
• Substrate binds cleft formed by aa of enzyme
• Functional groups of enzyme, also cofactors bond to
substrate, perform the catalysis;
Fig. 8.4
B. Binding site specificity
Substrate binding site is highly specific
• ‘Lock-and-key’ model: 3D shape ‘recognizes
substrate (hydrophobic, electrostatic, hydrogen bonds)
• ‘Induced-fit’ model: enzyme conformational change
after binding substrate
• galactose differs from
glucose, needs separate
galactokinase
Fig. 8.5 glucokinase
Glucokinase conformational change
Conformation change of
glucokinase on binding glucose
• Binding positions substrate to
promote reactions
• Large conformational change
adjusts actin fold, and facilitates
ATP binding
• Actin fold named for G-actin
(where first described; Fig. 7.8)
Fig. 8.6 glucokinase
(Yeast hexokinase)
Transition state complex
Energy Diagram: substrates are activated to react:
Activation energy: barrier to spontaneous reaction
Enzyme lowers activation energy
Transition-state complex is stabilized by diverse interactions
Fig. 8.7
Transition-state complex
Transition-state complex binds enzyme tightly:
• transition-state analogs are potent inhibitors of
enzymes (more than substrate analogs)
• make prodrugs that convert to active analogs at site
of action
• Abzymes: catalytic antibodies that have aa in
variable region like active site of transition enzyme:
•
•
Artificial enzymes: catalyze reaction
Ex. Abzyme to Cocaine esterase destroys cocaine in body
II. Catalytic mechanism of chymotrypsin - example enzyme
Chymotrypsin, serine protease, digestive enzyme:
• Hydrolyzes peptide bond (no reaction without enzyme)
• Serine forms covalent intermediate
• Unstable oxyanion (O-) intermediate
• Cleaved bond is
scissile bond
Fig. 8.8
B. Catalytic mechanism of chymotrypsin
1. Specificity of
binding:
Tyr, Phe, Trp on
denatured proteins
Oxyanion tetrahedral
intermediate
His57, Ser195, Asp
2. acyl-enzyme
intermediate
3. Hydrolysis of
acyl-enzyme
intermediate
Fig. 8.9
Mechanism of chymotrypsin, cont.
3. Hydrolysis of acylenzyme intermediate
• Released peptide
product
• Restores enzyme
Fig. 8.9
Energy diagram revisited with detail
Chymotrypsin reaction has several transitions:
• See several steps
• Lower energy barrier to uncatalyzed
Fig. 8.10
III. Functional groups in catalysis
Functional groups in catalysis:
• All enzymes stabilize transition state by electrostatic
• Not all enzymes form covalent intermediates
• Some enzymes use aa of active site (Table 1):
•
•
•
Ser, Lys, His - covalent links
His - acid-base catalysis
peptide backbone – NH stabilize anion
• Others use cofactors (nonprotein):
• Coenzymes (assist, not active on own)
• Metal ions (Mg2+, Zn2+, Fe2+)
• Metallocoenzymes (Fe2+-heme)
Coenzymes assist catalysis
Activation-transfer coenzymes:
• Covalent bond to part of
substrate; enzyme completes
• Other part of coenzyme binds to
the enzyme
• Ex. Thiamine pyrophosphate is
derived from vitamin thiamine;
• works with many different enzymes
• enzB takes H from TPP; carbanion
attacks keto substrate, splits CO2
Fig. 8.11
Other activation-transfer coenzymes
Activation-transfer coenzymes:
• Specific chemical group binds enzyme
• Other functional group participates directly in reaction
• Depends on enzyme for specificity of substrate, catalysis
Fig. 8.12 A
CoA forms thioesters with
many acyl groups:
acetyl, succinyl, fatty acids
Oxidation-reduction coenzymes
Oxidoreductase enzymes use other coenzymes:
•
•
•
•
Oxidation is loss of electrons (loss H, or gain O)
Reduction is gain electrons (gain H, loss of O)
Redox coenzymes do not form covalent bond to substrate
Unique functional groups
NAD+ (and FAD) special
role for ATP generation:
Ex. Lactate dehydrogenase
• oxidizes lactate to pyruvate
• transfers e- & H: to NAD+
-> NADH
Fig. 8.13 lactate dehydrogenase
Metal ions assist in catalysis
Positive metal ions attract electrons: contribute
• Mg2+ often bind PO4, ATP; ex. DNA polymerases
• Some metals bind anionic substrates
Fig. 8.14
ADH alcohol dehydrogenase
• oxidizes alcohol to acetaldehyde
and NAD+ to NADH
• Zn2+ assists with NAD+
(In Lactate dehydrogenase, a His
residue assisted the reaction)
pH affects enzyme activity
Each enzyme has characteristic pH optimum:
• Depends on active-site amino acids
• Depends on H bonds required for 3D structure
• Each enzyme has optimum
temperature for activity:
Humans 37oC
Taq polymerase
for PCR: 72oC
Fig. 8.15 optimal pH for enzyme
V. Mechanism-based inhibitors
Inhibitors decrease rate of enzyme
reaction:
• Mechanism-based inhibitors mimic
or participate in intermediate step of
reaction;
• Covalent inhibitors
• Transition-state analogs
• Heavy metals
Fig. 8.2 organophosphate inhibitors include
two insecticides, and nerve gas Sarin
Covalent inhibitors
Covalent inhibitors form covalent or very tight
bonds with functional groups in active site:
Fig. 8.16 DFP di-isopropylfluorophosphate prevents
acetylcholinesterase from degrading acetylcholine
Transition state analogs
Transition-state analogs bind
more tightly to enzyme than
substrate or product:
• Penicillin inhibits glycopeptidyl
transferase, enzyme that
synthesizes cross-links in
bacterial cell wall.
• Kills growing cells by inactivating
enzyme
Fig. 8.17 penicillin
Allopurinol treats gout
Allopurinol is suicide inhibitor of xanthine oxidase:
• Treatment for gout (decreases formation of urate)
Fig. 8.18
Basic reactions and classes of enzymes
6 basic classes of enzymes:
• Oxidoreductases
•
Oxidation-reduction reactions (one gains, one loses e-)
• Transferases
•
Group transfer – functional group from one to another
• Hydrolases cleave C-O, C-N and C-S bonds
•
addition of H2O in form of OH- and H+
• Lyases diverse cleave C-C, C-O, C-N
• Isomerases rearrange, create isomers of starting
• Ligases synthesize C-C, C-S, C-O and C-N bonds;
•
Reactions often use cleavage of ATP or others
Some example enzymes
Example enzymes:
Group transfer
– transamination
transfer of amino group
Isomerase
– rearranges atoms
ex. In glycolysis
Fig. 8.19
Key concepts
• Enzymes are proteins (or RNA) that are catalysts
• accelerate rate of reaction
• Enzymes are very specific or substrate
• Enzymes lower energy of activation – to reach highenergy intermediate state
• Functional groups at active site (amino acid
residues, metals, coenzymes) cause catalysis
• Mechanisms of catalysis include: acid-base,
formation covalent intermediates, transition state
stabilization
Review questions
4. The reaction shown fits into which classification?
a. Group transfer
b. Isomerization
c. Carbon-carbon bond breaking
d. Carbon-carbon bond formation
e. Oxidation-reduction
5. The type of enzyme that catalyzes
this reaction is which of the following?
a. Kinase
b. Dehydrogenase
c. Glycosyltransferase
d. Transaminase
e. isomerase