Post on 19-Mar-2016
description
Enzyme Catalysis
Prof. Dr. Supartono, Prof. Dr. Supartono, M.SM.S
PostgraduateSemarang University State
Objective
To understand how enzymes work at the molecular
level.
Ultimately requires total structure determination, but can learn much through biochemical
analysis.
To Be Explained
• Specificity– For specific substrates– Amino acids residues involved
• Catalysis– Amino acids involved– Specific role(s)
• Regulation
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EnzymesEnzymes• Enzymes are catalysts. They increase the speed of a chemical reaction without themselves undergoing any permanent chemical change.
• Enzymes are neither used up in the reaction, nor do they appear as reaction products.
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Discovery of Enzymes• 1825 Jon Jakob Berzelius discovered 1825 Jon Jakob Berzelius discovered
the catalytic effect of enzymes.the catalytic effect of enzymes.• 1926 James Sumner isolated the 1926 James Sumner isolated the
first enzyme in pure form.first enzyme in pure form.• 1947 Northrup and Stanley 1947 Northrup and Stanley
together with Sumner were together with Sumner were awarded the Nobel prize for the awarded the Nobel prize for the isolation of the enzyme isolation of the enzyme pepsinpepsin..
Berzelius
Sumner
NorthrupStanley24/04/23 5Supartono
Enzyme Characteristics• High molecular weight High molecular weight proteins proteins with masses with masses ranging from 10,000 to ranging from 10,000 to as much as 2,000,000 as much as 2,000,000 grams per molegrams per mole
• Substrate specific Substrate specific catalystscatalysts
• Highly efficienHighly efficien, , increasing reaction increasing reaction rates by a factor as rates by a factor as high as 10high as 1088
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Enzyme Nomenclature• The earliest enzymes that were discovered have common names:
i.e. Pepsin, Renin, Trypsin, Pancreatin
• The enzyme name for most other enzymes ends in “ase”
• The enzyme name indicates the substrate acted upon and the type of reaction that it catalyzes24/04/23 7Supartono
Enzyme NamesExamples of Enzyme Names• Glutamic Oxaloacetic Transaminase (GOT)
• L-aspartate: 2-oxoglutarate aminotransferase.
Enzyme names tend to be long and complicated. They are often abbreviated with acronyms24/04/23 8Supartono
Enzyme Mechanisms Enzymes lower the activation energy for reactions and shorten the path from reactants to products
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Enzyme Mechanisms The basic enzyme reaction can be represented as follows:
E + S ES E + PEnzyme Substrate Enzyme substrate Enzyme Product(s) complex
The enzyme binds with the substrate to form the Enzyme-Substrate Complex. Then the substrate is released as the product(s).24/04/23 10Supartono
Enzyme MechanismsDiagram of the action of the enzyme sucrase on sucrose.
E+SES E+P
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Enzyme Specificity• The action of an enzyme depends primarily on the tertiary and quaternary structure of the protein that constitutes the enzyme.
• The part of the enzyme structure that acts on the substrate is called the active site.
• The active site is a groove or pocket in the enzyme structure where the substrate can bind.24/04/23 12Supartono
Cofactors• Cofactors are other compounds or ions that enzymes require before their catalytic activity can occur.
• The protein portion of the enzyme is referred to as the apoenzyme.
• The enzyme plus the cofactor is known as a holoenzyme.
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CofactorsCofactors may be one of three types1. Coenzyme: A non protein organic
substance that is loosely attached to the enzyme
2. Prosthetic Group: A non protein organic substance that is firmly attached to the enzyme
3. Metal ion activators: K+, Fe2+, Fe3+, Cu2+, Co2+, Zn2+, Mn2+, Mg2+, Ca2+, or Mo2+,
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Types of Cofactors
• Enzymes have varying degrees of specificity.
• One cofactor may serve many different enzymes. 1524/04/23 15Supartono
Types of Cofactors
• Enzymes have varying degrees of specificity.
• One cofactor may serve many different enzymes.24/04/23 16Supartono
Enzymes and Cofactors
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Factors Affecting Enzyme Activity
Enzymes and Reaction Rates
Factors that influence reaction rates of Enzyme catalyzed reactions include1.Enzyme and substrate concentrations
2.Temperature3.pH
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Enzymes and Reaction Rates• At low concentrations,
an increase in substrate concentration increases the rate because there are many active sites available to be occupied
• At high substrate concentrations the reaction rate levels off because most of the active sites are occupied
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Substrate concentration• The maximum velocity of a reaction is reached when the active sites are almost continuously filled.
• Increased substrate concentration after this point will not increase the rate.
• Vmax is the maximum reaction rate24/04/23 21Supartono
Substrate concentration• Vmax is the maximum reaction rate
• The Michaelis-Menton constant , Km is the substrate concentration when the rate is ½ Vmax
• Km for a particular enzyme with a particular substrate is always the same
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Effect of Temperature• Higher temperature increases the number of effective collisions and therefore increases the rate of a reaction.
• Above a certain temperature, the rate begins to decline because the enzyme protein begins to denature
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Effect of pH• Each enzyme has an optimal pH at which it is most efficient
• A change in pH can alter the ionization of the R groups of the amino acids.
• When the charges on the amino acids change, hydrogen bonding within the protein molecule change and the molecule changes shape.
• The new shape may not be effective.Pepsin is most efficient
at pH2.5-3 while Trypsin is efficient at a much higher pH
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pH
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Effects of pH on Enzyme Activity
• Binding of substrate to enzyme• Ionization state of “catalytic” amino acid residue side chains
• Ionization of substrate• Variation in protein structure
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Temperature
RelativeActivity
ba
Temperature
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Inhibitors
• Covalent– Reversible– Irreversible
• Non-covalent: reversible
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Enzyme Binding Sites
• Active Site = Binding Site + Catalytic Site
• Regulatory Site: a second binding site, occupation of which by an effector or regulatory molecule, can affect the active site and thus alter the efficiency of catalysis – improve or inhibit.
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Active Site
Binding and Catalysis
General Characteristics
• Three dimensional entity• Occupies small part of enzyme volume
• Substrates bound by multiple weak interactions
• Clefts or crevices• Specificity depends on precise arrangement of atoms in active site
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Models
• Lock and Key Model: the active site exists “pre-formed” in the enzyme prior to interaction with the substrate.
• Induced Fit Model: the enzyme undergoes a conformational change upon initial association with the substrate and this leads to formation of the active site.
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Enzyme Mechanics
• An enzyme-substrate complex forms when the enzyme’s active site binds with the substrate like a key fitting a lock.
• The shape of the enzyme must match the shape of the substrate.
• Enzymes are therefore very specific; they will only function correctly if the shape of the substrate matches the active site.
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Induced Fit Theory
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Induced Fit Theory• The substrate molecule normally does not fit exactly in the active site.
• This induces a change in the enzymes conformation (shape) to make a closer fit.
• In reactions that involve breaking bonds, the inexact fit puts stress on certain bonds of the substrate.
• This lowers the amount of energy needed to break them.24/04/23 35Supartono
Induced Fit Theory• The enzyme does not actually form a chemical bond with the substrate. After the reaction, the products are released and the enzyme returns to its normal shape.
• Because the enzyme does not form chemical bonds with the substrate, it remains unchanged.
• The enzyme molecule can be reused repeatedly
• Only a small amount of enzyme is needed
36
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Identification and Characterization of Active
Site
• Structure: size, shape, charges, etc.
• Composition: identify amino acids involved in binding and catalysis.
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Binding or Positioning Site(Trypsin)
NH CH C NHO
N C
complementary binding or posit ioning site
"SPECI FI CI TY"_
+
arginine or lysine
"long + side chain"
H2O
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Binding or Positioning Site(Chymotrypsin)
NH CH C NHO
N C
"aromatic side chain"
"SPECI FI CI TY"
complementary binding or posit ioning site
phenylalaninetyrosinetryptophan
Hydrophobic Pocket
H2O
O
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Catalytic Site(e.g. Chymotrypsin)
NH CH C NHO
N C
catalyt ic sitecomplementary
"CATALYSI S"
H2O
O
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Probing the Structure of the Active Site
Model Substrates
Model Substrates(Chymotrypsin)
H2O(ROH)
NH CH CN
R
NHO
C
acyl transfer to H2Oaromaticside chain
peptide bond
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Peptide Chain?
All Good Substrates!
H3N CH C NH
O
C
R
NH CH CH3N
R
NH2
O
(or -OCH3)
or
H3N CH C
R
NH2
O(or -OCH3)
or
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-amino group?
Good Substrate!
H2C C NH2
O
R (OCH3)
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Side Chain Substitutions
Good Substrates
Cyclohexyl
t-butyl-
CH3
CH3
CH3
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ConclusionBulky Hydrophobic Binding Site
CH C XO
Y
"Hydrophobic Acyl Group Transferase"
= hydrophobic posit ioning group
X,Y = various
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Probing the Structure of the Active Site
Competitive Inhibitors
Arginase
H2N
C
NH
(CH2)3
CH COOH3N
NH2H2O
NH3
(CH2)3
CH COOH3N
H2N
C
O
NH2
+
+
-+
ureaornithinearginine
+ -
+
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Good Competitive Inhibitors
NH3
NH
NH3
(CH2)3
CH COOH3N
NH3
(CH2)4
CH COOH3N
O
(CH2)2
CH COOH3N
CH
NH2
-+(
ornithine
(+ -
++
+
-
(
canavaninelysine
+
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Poor Competitive Inhibitors
All Three Charged Groups are Important
NH3
(CH2)3CH2H3N
NH3
(CH2)3H2C COO
CH3
(CH2)3CH COOH3N
+ -
++
+ -
a-aminovaleric acid putrescine(l,4-diaminobutane)
4-aminovaler ic acid
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ConclusionActive Site Structure of Arginase
+-
-bindingsite
catalytic site
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Identifying Active Site Amino Acid Residues
Covalent Inactivation
Diisopropyl Phosphofluoridate
Inactivates Chymotrypsin by forming a 1:1 covalent adduct to Serine195.
Iodoacetic acid inactivates Ribonuclease by reacting with His12
and His119.
CH O P O CH
CH3
CH3
CH3
CH3
F
O
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Affinity Labeling(General Approach)
Positioning Group
Reactive Group
YBinding Site
+
XX
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Affinity Labeling(Tosyl-L-phenylalanine chloromethylketone)
Inactivates Chymotrypsin by forming a 1:1 covalent adduct to
Histidine57
O S O
NH
CHCH2
CH3
ReactiveGroup
PositioningGroup
O
C
O
CH2 Cl
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Trapping of Enzyme-Bound Intermediate(Chymotrypsin)
Implicates Ser195 in catalytic mechanism.
CT CH2 OH O2N O O C CH3
O
Ser195
O2N
CT CH2 O C CH3
O
O
"acyl" enzyme stable at pH 3
p-nitrophenylacetate
+
O–
p-nitrophenol
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Catalytic Mechanisms
Mechanisms of Catalysis
• Acid-base catalysis• Covalent catalysis• Metal ion catalysis• Proximity and orientation effects• Preferential binding (stabilization) of the transition state
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Acid-Base Catalysis
Keto-Enol Tautomerization
R C CH3
O
R C
OH
CH2
Ketone Enol
Uncatalyzed Reaction
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General Acid Catalysis
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General Base Catalysis
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General Acids
COOH NH3
CH2 OH
CH2 OH
SH
HN NH
CH2
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General Bases
COO NH2
SHN N:
CH2
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Figure 11-10
Ribonuclease A
N
N
O
O
OH
O
O
CH2
O
PO O
O
N
N
N
N
O
NH2
OH
CH2OPO
O
O
PO O
O
Adenosine
UridineRibonuclease A
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Figure 11-10 part 1
Mechanism of RNase A
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Figure 11-10 part 2
Mechanism of RNase A
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Covalent Catalysis(Principle)
Slow
H2O + A–B ——> AOH + BH
A-B + E-H ——> E-A + BH
E-A + H2O ——> A-OH + E-H
Fast
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Covalent Catalysis(Chymotrypsin)
CT CH2 OH O2N OO C CH3
O
CT CH2 O C CH3
O
CT CH2 OH
Ser195
O OO2N
CT CH2 O C CH3
O
HO C CH3
O
"rate limiting"++ H2O
+
f ast+
fast
NOTE: New Reaction Pathway24/04/23 69Supartono
Metal Ion Catalysis
• Metalloenzymes: contain tightly bound metal ions for catalytic activity
• Metal-activated enzymes: loosely bound metal ions from solution
• Charge stabilization• Water ionization• Charge shielding24/04/23 70Supartono
Metalloenzymes
• Catalytically essential (tightly bound): Fe2+, Fe3+, Cu2+, Mn2+, and Co2+
• Structural metal ions: Na+, K+, and Ca2+
• Both: Mg2+ and Zn2+
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Proximity and Orientation Effects
Rate of a reaction depends
on:
• Number of collisions• Energy of molecules• Orientation of molecules• Reaction pathway (transition state)
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Proximity
V = k[A][B]
[A] and [B] = ~13M on enzyme surface
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Page 336
Biomolecular Reaction of Imidazole with p-Nitrophenylacetate
(Intermolecular)
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Page 336
Intramolecular Reaction of Imidazole with p-Nitrophenylacetate
(Intramolecular)
Intramolecular Rate = 24x Intermolecular Rate24/04/23 75Supartono
Orientation
A BBA
C
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Figure 11-14
Geometry of an SN2 Reaction
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Stabilize Transition State
HO
R
Br
Y
R
R
Br
Y
R
Z
_
"strain" "stabilized"
HO C
R
XX
CR
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Page 338
Steric Strain in Organic Reactions
Reaction Rate: R=CH3 is 315x vs R=H24/04/23 79Supartono
Figure 11-15
Effect of PreferentialTransition State Binding
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Transition State Analogs
Powerful Enzyme Inhibitors
Page 339
Proline Racemase(planar transition state)
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Page 339
Transition State Analogs of Proline
Binding = 160x versus Proline24/04/23 83Supartono
Serine Proteases
ChymotrypsinTrypsinElastase
etc.
Kinetic Analysis of Chymotrypsin(Hydrolysis of p-nitrophenylacetate)
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Mechanism of Chymotrypsin
CT CH2 OH O2N O C CH3
O
CT CH2 O C CH3
O
CT CH2 OH
OO2N
CT CH2 O C CH3
O
HO C CH3
O
"rate limiting"++ H2O
+fast+
fast
p-nitrophenolat
e
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Identification of the Catalytic Residues
(Reaction of Chymotrypsin with DIPF)
Special Reactivity of Serine195
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Identification of the Catalytic Residues
(Reaction of Chymotrypsin with TPCK)
Affinity Labeling24/04/23 88Supartono
X-Ray Structure of Bovine Trypsin(Ribbon Diagram)
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Figure 11-26
Active Site Residues of Chymotrypsin(Catalytic Triad)
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Figure 11-29 part 1
Catalytic Mechanism of the Serine Proteases
Catalytic Triad
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Figure 11-29 part 2
Catalytic Mechanism of the Serine Proteases
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Figure 11-29 part 3
Catalytic Mechanism of the Serine Proteases
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Figure 11-29 part 4
Catalytic Mechanism of the Serine Proteases
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Figure 11-29 part 5
Catalytic Mechanism of the Serine Proteases
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Figure 11-29 part 5
Catalytic Mechanism of the Serine Proteases
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Figure 11-30a
Transition State Stabilization in the Serine
Proteases
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Figure 11-30b
Transition State Stabilization in the Serine
Proteases
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Mechanism of Chymotrypsin
CT CH2 OH O2N O C CH3
O
CT CH2 O C CH3
O
CT CH2 OH
OO2N
CT CH2 O C CH3
O
HO C CH3
O
"rate limiting"++ H2O
+fast+
fast
p-nitrophenolat
e
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