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Lecture 17
– Exams in Chemistry office with M’Lis.
Please show your ID to her to pick up
your exam.
– Quiz on Friday
– Enzyme mechanisms
General Acid-Base Catalysis
• General acid catalysis- a process in which partial proton
transfer from a Brønstead acid (a species that can donate
protons) lowers the free energy of a reaction’s transition
state.
• General base catalysis - process in which partial proton
abstraction by a Brønstead base (a species that can
combine with a proton) lowers the free energy of a
reaction’s transition state.
• General acid-base catalysis-a combination of both.
Page 497
Figure 15-1a Mechanisms of keto–enol tautomerization.
(a) Uncatalyzed.
Page 497
Figure 15-1bMechanisms of keto–enol tautomerization.
(b) General acid catalyzed.
Page 497
Figure 15-1c Mechanisms of keto–enol
tautomerization.
(c) General base catalyzed.
General Acid Base Catalysis
• Ex. Ester hydrolysis
O
C
d
H
H
d
O
+ H+
OR
C
H2O
d
OR
C OR
O+
H
H
H+
O
C
O
O
- H+
OH
C
+ ROH
O
H
OR
H+
General Acid-Base Catalysis
• Large number of possible amino acids
• Requires that they can accept and donate a proton
• Glu, Asp
• Lys, His, Arg
• Cys, Ser, Thr
• Also can include metal cofactors
• Example can be observed in carboxypeptidase A (both acid
and base catalysis)
General Acid-Base Catalysis
• Ex. Carboxypeptidase A
Zn plays role of acid (4th ligand
is normally H2O, but it is
displaced by peptide binding)
Glu72
His196
His69
O
Zn++
H O


d
d
H
Glu270 C-OGlu acts as base catalyst
to polarize water and
form nucleophile
R
H-C
N
 d
dO
C
+ Arg145
CO2H
Key aas that holds
molecule in place
HO-Tyr248
H-C-R
NH
C
Tyr also plays role
as 2nd acid catalyst
O
+ Arg
Study of Enzyme Mechanisms
• X-ray crystallography-crystallize the molecule with
substrate in place and compare to crystal structure of the
molecule without the substrate (differences in structure)
• For carboxypeptidase A they could show that
• Water is expelled by binding of substrate
• Arg145 moves 2Å closer to the carboxyl group of the
substrate
• Glu270 moves 2Å towards the C=O group
• Tyr248 moves 12Å towards the amide plane of the
peptide
• Also able to show what aa surround certain groupsTyr248 in a hydrophobic pocket.
Study of Enzyme Mechanisms
• Check the pH profile of the enzyme.
• For carboxypeptidase
• The coordination of Zn by His69 and His196 (pK 6.0)
•
Tyr248 (pK 9.1)
Example in
book: RNAse
(p. 499)
8.5
6.7
Log (Vmax/KM)
6
7
pH
8
9
Lysozyme (Strain and Acid Catalysis)
• Hen Egg White (HEW) Lysozyme digests bacterial cell
walls.
• Cleaves (1 4) glycosidic linkages from N-acetylmuramic
acid (NAM) to N-acetylglucosamine (NAG)
• Requires about 6 sugars for good recognition.
SugA-SugB-SugC-SugD-SugE-SugF
Lysozyme (Strain and Acid Catalysis)
• In theory
SugA-SugB-SugC-SugD-SugE-SugF
Asp52
-O
d
O
E
O
D
:
O
H+
C
O
OH
OH
• Must distort ring into a flat, planar shape
• Supply acid catalysis
O
C
OR
OH
O
C
OH
Glu35
Lysozyme (Strain and Acid Catalysis)
• In practice
Page 510
Figure 15-12 Interactions of lysozyme with its substrate.
Page 510
Figure 15-11 Chair and halfchair conformations.
Study of Enzyme Mechanisms
•
•
•
•
•
•
•
•
Lysozyme
Only the D ring is strained
Glu35 is in a hydrophobic environment
Asp52 is in a hydrophilic environment
Covalent modification of the active site
Block essential groups
May or may not act at active site
Cd or R-As=O (trivalent arsenic)
The Aspartate Proteases
• Pepsin, Renin, HIV protease (AZT targets this)
• General acid-base catalysis
Serine hydrolases: trypsin, chymotrypsin,
elastase
• Synthesized in pancreas as inactive zymogen (ex.
trypsinogen)
• Generally operate by "charge relay system"
• Asp102, His57, Ser195 conserved in all 3 enzymes.
Asp102
COO
His57
Ser195
O
H N
NH
R'
C
H
N
R H
O
1
Serine hydrolases: trypsin, chymotrypsin,
elastase
Asp102
His57
COO H
2
Ser195
O
R'
NH
C
:
N
N
R H
Asp102
COO
Rate
limiting
step for
amides
O-
His57
Ser195
H N
O
NH
R'
C
O
RNH2
Serine hydrolases: trypsin, chymotrypsin,
elastase
Asp102
COO
His57
Ser195
-
O
H N
NH
Asp102
C
H
H
His57
O
O
Ser195
COO H
O
N
R'
NH
3
R'
C
O
H
O-
Rate
limiting
step for
esters
Serine hydrolases: trypsin, chymotrypsin,
elastase
Asp102
COO
His57
Ser195
H N
O
H
NH
R'
C
O
H
O
Charge-relay systems
• Relay charges between amino acid side chains in order to
catalyze the reaction.
Summary: various methods to increase rate
• Increase frequency of the correct group in the correct place
e.g. proximity effect
• Lower EA by specific catalysis -acid-base catalysis,
nucleophile or electrophile
• Raise energy of reactants (closer to EA) - ring distortion,
transition state analog
• Provide alternate low EA pathway - covalent catalysis.]
• Michaelis Menten
• Lineweavear Burk
• Eadie Hofstee
• Competitive inhibition
• Noncompetitive inhibition
Terms to review for enzymes
•
•
•
•
•
•
•
•
•
Cofactor
Coenzyme
Prosthetic group
Holoenzyme
Apoenzyme
Lock and Key
Transition analog model
Induced fit
Active site, binding site, recognition site, catalytic site
Catalytic Mechanisms
• Acid-base catalysis
• Covalent catalysis
• Metal ion catalysis
• Proximity and orientation effects (ex. anhydride)
• Preferential binding of the transition state complex
General Acid-Base Catalysis
• Large number of possible amino acids
• Requires that they can accept and donate a proton
• Glu, Asp
• Lys, His, Arg
• Cys, Ser, Thr
• Also can include metal cofactors (metal ion catalysis)
• Example can be observed in RNAse
Page 499
Figure 15-2 The pH dependence of V¢max/K¢M in the
RNase A–catalyzed hydrolysis of cytidine-2¢,3¢ -cyclic
phosphate.
Example in
book: RNAse
(p. 499)
RNAse mechanism
•His12 acts as general
base-takes proton from
RNA 2’-OH-making a
nucleophile which
attacks the phosphate
group.
•His119 acts as a
general acid to promote
bond scission.
Page 499
•2’,3’ cyclic intermediate
is hydrolyzed through
the reverse of the first
step-water replaces the
leaving group. His12 is
the acid, His119 acts
as the base
Covalent catalysis
• Rate acceleration through the transient formation of a
catalyst-substrate covalent bond.
• Example-decarboxylation of acetoacetate by primary
amines
• Amine nucleophilically attacks carbonyl group of
acetoacetate to form a Schiff base (imine bound)
Figure 15-4 The decarboxylation of acetoacetate.
Page 500
uncatalyzed
e- sink
Catalyzed by primary amine
Covalent catalysis
• Made up of three stages
1. The nucleophilic reaction between the catalyst and
the substrate to form a covalent bond.
2. The withdrawal of electrons from the reaction center
by the now electrophilic catalyst
3. The elimination of the catalyst (reverse of 1.)
• Nucleophilic catalysis - covalent bond formation is
limiting.
• Electrophilic catalysis-withdrawal of electrons is rate
limiting
Covalent catalysis
• Nucleophilicity is related to basicity. Instead of
abstracting a proton, nucleophilically attacks to make
covalent bond.
• Good covalent catalysts must have high nucleophilicity
and ability to form a good leaving group.
• Polarized groups (highly mobile e-) are good covalent
catalysts: imidazole, thiols.
• Lys, His, Cys, Asp, Ser
• Coenzymes: thiamine pyrophosphate, pyridoxal
phosphate.
Covalent Catalysis
• Form transient, metastable intermediates that can supply
bond energy into the reaction.
Examples
structures
Side chain
Chymotrypsin
NH
O
Trypsin
Serine
Elastase
RC-O-CH -CH
2
(acyl ester) COO-
Serine
O
NH
-O-P-O-CH -CH
2
O
(phosphoryl ester)
COO-
acetylcholinesterase
Phosphoglucomutase
Alkaline phosphatase
Covalent Catalysis
Group
O
Cysteine
Examples
structures
NH
Papain
3-PGAL-DH
RC-S-CH2-CH
(acyl cysteine) COO-
Histidine
O
-O-P-N
CH
NH
O
(phosphoryl imidazole)
COO-
Succinate thiokinase
Covalent Catalysis
Group
R'
Lysine
Examples
structures
NH
R-C=N-(CH2)4-CH
(Schiff base)
COO-
Aldolase
Transaldolase
Metal ion catalysis
• Almost 1/3 of all enzymes use metal ions for
catalytic activity. 2 main types:
1. Metalloenzymes-have tightly bound metal ions, mmost
commonly transition metal ions such as Fe2+, Fe3+, Cu2+,
Zn2+, Mn2+, or Co3+
2. Metal-activated enzymes-loosely bind metal ions form
solution-usually alkali or alkaline earth metals-Na+, K+,
Ca2+
Metal ion catalysis
• Three ways for catalysis
1. Binding to substrates to orient them properly for the
reaction
2. Mediating oxidation-reduction reactions through
reversible changes in the metal ion’s oxidation state
3. Electrostatically stabilizing or shielding negative charges.
Serine Hydrolases (Proteases)
• Chymotrypsin, trypsin and elastase.
• All have a reactive Ser necessary for activity.
• Catalyze the hydrolysis of peptide (amide) bonds.
• Chymotrypsin can act as an esterase as well as a
protease.
• Study of esterase activity provided insights into the
catalytic mechanism.
O
CH3 C
O
NO2
p-Nitrophenylacetate
Chymotrypsin
H2O
2H+
O
CH3 C
Acetate
O-
+
-O
NO2
p-Nitrophenolate
Serine Hydrolases (Proteases)
• Reaction takes place in 2 phases
1. The “burst phase”-fast generation of pnitrophenolate in stoichiometric amounts with
enzyme added
2. The “steady-state phase”-p-nitrophenolate
generated at reduced but constant rate; independent
of substrate concentration.
Page 516
Figure 15-18
Time course of pnitrophenylacetate hydrolysis as catalyzed by
two different concentrations of chymotrypsin.
O
CH3 C
O
NO2
+ Enzyme
Chymotrypsin
p-Nitrophenylacetate
-O
FAST
O
CH3 C
NO2
p-Nitrophenolate
O-Enzyme
Acyl-enzyme intermediate
SLOW
O
H2O
2H+
CH3 C O- + Enzyme
Acetate
Chymotrypsin
• Follows a ping pong bi bi mechanism.
• Rate limiting step for ester hydrolysis is the deacylation
step.
• Rate limiting step for amide hydrolysis is first step (enzyme
acylation).
Identification of catalytic residues
(active Ser)-CH2OH
+
• Identified catalytically
important residues by
chemical labeling
studies.
• Ser195-identified
using
diisopropylphosphofluoridate (DIPF)
• Irreversible!
CH(CH3)2
O
F-P=O
Diisopropylphospho
-fluoridate (DIPF)
O
CH(CH3)2
CH(CH3)2
O
(active Ser)-CH2O-P=O
O
CH(CH3)2
DIP-enzyme
Identification of catalytic residues
• His57 was identified through affinity labeling
• Substrate analog with a reactive group that specifically
binds to the active site of the enzyme forms a stable
covalent bond with a nearby susceptible group.
• Reactive substrate analogs are sometimes called “Trojan
horses” of biochemistry.
• Affinity labeled groups can be identified by peptide
mapping.
• For chymotrypsin, they used an analog to Phe.
Identification of catalytic residues
O
CH3
S
CH2 O
NH CH
C
CH2Cl
O
Tosyl-L-phenylalanine chloromethyl ketone (TPCK)
Page 517
Figure 15-19 Reaction of TPCK with
chymotrypsin to alkylate His 57.
Homology among enzymes
• Bovine chymotrypsin, bovine trypsin and porcine
elastase are highly homologous
• ~40% identical over ~240 residues.
• All enzymes have active Ser and catalytically essential
His
• X-ray structures closely related.
• Asp102 buried in a solvent inaccessible pocket (third
enzyme in the “catalytic triad”)
X-ray structures explain differences in
substrate specificity
• Chymotrypsin - bulky aromatic side chains (Phe, Trp, Tyr)
are preferred and fit into a hydrophobic binding pocket
located near catalytic residues.
• Trypsin - Residue corresponding to chymotrypsin Ser189
is Asp (anionic). The cationic side chains of Arg and Lys
can form ion pairs with this residue.
• Elastase - Hydrolyzes Ala, Gly and Val rich sequences.
The specificity pocket is largely blocked by side chains of
Val and a Thr residue that replace Gly residues that line the
binding pocket of chymotrypsin and trypsin.
X-Ray structure of
bovine trypsin.
(a) A drawing of the enzyme in complex.
Page 518
Figure 15-20a
Page 519
Figure 15-20b
X-Ray structure of bovine
trypsin. (b) A ribbon diagram of trypsin.
Page 519
Figure 15-20c
X-Ray structure of bovine
trypsin. (c) A drawing showing the surface of trypsin
(blue) superimposed on its polypeptide backbone
(purple).
Page 520
Figure 15-21 The active site
residues of chymotrypsin.
Page 521
Figure 15-22 Relative positions of the active site residues in
subtilisin, chymotrypsin, serine carboxypeptidase II, and
ClpP protease.
Page 522
Figure 15-23
Catalytic
mechanism of
the serine
proteases.