Transcript Lecture 9

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
Terms to review for enzymes
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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
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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.
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•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.
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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.
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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)
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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.
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Figure 15-20a
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Figure 15-20b
X-Ray structure of bovine
trypsin. (b) A ribbon diagram of trypsin.
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Figure 15-20c
X-Ray structure of bovine
trypsin. (c) A drawing showing the surface of trypsin
(blue) superimposed on its polypeptide backbone
(purple).
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Figure 15-21 The active site
residues of chymotrypsin.
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Figure 15-22 Relative positions of the active site residues in
subtilisin, chymotrypsin, serine carboxypeptidase II, and
ClpP protease.
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Figure 15-23
Catalytic
mechanism of
the serine
proteases.