The Organic Chemistry of Enzyme Catalyzed Reactions Revised ...
Transcript of The Organic Chemistry of Enzyme Catalyzed Reactions Revised ...
The Organic Chemistry of Enzyme-Catalyzed Reactions
Revised Edition
Professor Richard B. SilvermanDepartment of Chemistry
Department of Biochemistry, Molecular Biology, and Cell BiologyNorthwestern University
The Organic Chemistry of Enzyme-Catalyzed Reactions
Chapter 1
Enzymes as Catalysts
For published data regarding any enzyme see:http://www.brenda-enzymes.info/Nomenclature Enzyme Names EC Number Common/ Recommended Name Systematic Name Synonyms CAS Registry Number
Reaction & Specificity Pathway Catalysed Reaction Reaction Type Natural Substrates and Products Substrates and Products Substrates Natural Substrate Products Natural Product Inhibitors Cofactors Metals/ Ions Activating Compounds Ligands
Functional Parameters Km Value Ki Value pI Value Turnover Number Specific Activity pH Optimum pH Range Temperature Optimum Temperature Range
Isolation & Preparation Purification Cloned Renatured Crystallization
Organism- related information Organism Source Tissue Localization
Stability pH Stability Temperature Stability General Stability Organic Solvent Stability Oxidation Stability Storage Stability
Enzyme Structure Sequence/ SwissProt link 3D-Structure/ PDB link Molecular Weight Subunits Posttranslational Modification
Disease & References Disease References
Application & Engineering Engineering Application
What are enzymes, and how do they work?
• First “isolation” of an enzyme in 1833• Ethanol added to aqueous extract of malt • Yielded heat-labile precipitate that was
utilized to hydrolyze starch to soluble sugar; precipitate now known as amylase
• 1878 - Kühne coined term enzyme - means “in yeast”
• 1898 - Duclaux proposed all enzymes should have suffix “ase”
• Enzymes - natural proteins that catalyze chemical reactions
• First enzyme recognized as protein was jack bean urease
• Crystallized in 1926• Took 70 more years (1995), though, to obtain
its crystal structure
• Enzymes have molecular weights of several thousand to several million, yet catalyze transformations on molecules as small as carbon dioxide and nitrogen
• Function by lowering transition-state energies and energetic intermediates and by raising the ground-state energy
• Many different hypotheses proposed for how enzymes catalyze reactions
• Common link of hypotheses: enzyme-catalyzed reaction always initiated by the formation of an enzyme-substrate (or ES) complex in a small cavity called the active site
• 1894 - Lock-and-key hypothesis - Fischer proposed enzyme is the lock into which the substrate (the key) fits
• Does not rationalize certain observed phenomena:
Compounds having less bulky substituents often fail to be substrates
Some compounds with more bulky substituents bind more tightly
Some enzymes that catalyze reactions between two substrates do not bind one substrate until the other one is bound
1958 - Induced-fit hypothesis proposed by Koshland:
When a substrate begins to bind to an enzyme, interactions induce a conformational change in the enzyme
Results in a change of the enzyme from a low catalytic form to a high catalytic form
Induced-fit hypothesis requires a flexible active site
Concept of flexible active site stated earlier by Pauling (1946):
Hypothesized that an enzyme is a flexible template that is most complementary to substrates at the transition state rather than at the ground state
Therefore, the substrate does not bind most effectively in the ES complex
As reaction proceeds, enzyme conforms better to the transition-state structure
Transition-state stabilization results in rate enhancement
• Only a dozen or so amino acid residues may make up the active site
• Only two or three may be involved directly in substrate binding and/or catalysis
Why is it necessary for enzymes to be so large?• Most effective binding of substrate results
from close packing of atoms within protein• Remainder of enzyme outside active site is
required to maintain integrity of the active site• May serve to channel the substrate into the
active siteActive site aligns the orbitals of substrates and
catalytic groups on the enzyme optimally for conversion to the transition-state structure-- called orbital steering
• Enzyme catalysis characterized by two features: specificity and rate acceleration
• Active site contains amino acid residues and cofactors that are responsible for the above features
• Cofactor, also called a coenzyme, is an organic molecule or metal ion that is essential for the catalytic action
Specificity of Enzyme-Catalyzed Reactions• Two types of specificity: (1) Specificity of binding
and (2) specificity of reactionSpecificity of Binding
• Enzyme catalysis is initiated by interaction between enzyme and substrate (ES complex)
• k1, also referred to as kon, is rate constant for formation of the ES complex
• k-1, also referred to as koff, is rate constant for breakdown of the complex
• Stability of ES complex is related to affinity of the substrate for the enzyme as measured by Ks, dissociation constant for the ES complex
Ks =
E + S E . E . E + Pk2
k-1
k1
k-1
k1
S P
Scheme 1.1
kon
koff
Michaelis complex
When k2 << k-1,k2 called kcat (turnover number)Ks called Km (Michaelis-Menten constant)
Generalized enzyme-catalyzed reaction
kcat represents the maximum number of substrate molecules converted to product molecules per active site per unit of time; called turnover number
Table 1.1. Examples of Turnover Numbersa
Enzyme Turnover numberkcat (s-1)
papain 10carboxypeptidase 102
acetylcholinesterase 103
kinases 103
dehydrogenases 103
aminotransferases 103
carbonic anhydrase 106
superoxide dismutase 106
catalase 107
aEigen, M.; Hammes, G.G. Adv. Enzymol. 1963, 25, 1.
• Km is the concentration of substrate that produces half the maximum rate
• Km is a dissociation constant, so the smaller the Km the stronger the interaction between E and S
• kcat/Km is the specificity constant - used to rank an enzyme according to how good it is with different substrates
Upper limit for is rate of diffusion (109 M-1s-1)Km
kcat
How does an enzyme release product so efficiently given that the enzyme binds the transition state structure about 1012 times more tightly than it binds the substrate or products?
After bond breaking (or making) at transition state, interactions that match in the transition-state stabilizing complex are no longer present.
Therefore products are poorly bound, resulting in expulsion.
As bonds are broken/made, changes in electronic distribution can occur, generating a repulsive interaction, leading to expulsion of products
E • S complexFigure 1.1Non-covalent interactions
electrostatic(ionic)
C
O
O
+
RNH3
ion -dipo le R
C NH3
R'
Oδ
δ+
δipole-δipole R
C O
R'
Oδδδ
δ
H
H-bonδing
O
RC O HO
H
chargetransfer
A
D
D
A
hyδrophobic
ORC
O
∆Gº = -RTlnKeq
If Keq = 0.01, ∆Gº of -5.5 kcal/mol needed to shift Keq to 100
Specific Forces Involved in E•S Complex Formation
Figure 1.2
NH3 O
OH
CH3
COCH2
CH2
NMe3
+
+
δ−
δ+
δ−
δipole-δipoleδ+
ion-δipole
O
O
ionic
Examples of ionic, ion-dipole, and dipole-dipole interactions. The wavy line represents the
enzyme active site
H-bonds
A type of dipole-dipole interaction between X-H and Y: (N, O)
Figure 1.3
H-bonds
Hydrogen bonding in the secondary structure of proteins: a-helix and b-sheet.
Charge Transfer Complexes
• When a molecule (or group) that is a good electron donor comes into contact with a molecule (or group) that is a good electron acceptor, donor may transfer some of its charge to the acceptor
Hydrophobic Interactions
• When two nonpolar groups, each surrounded by water molecules, approach each other, the water molecules become disordered in an attempt to associate with the water molecules of the approaching group
• Increases entropy, resulting in decrease in the free energy (DG = DH-TDS)
van der Waals Forces
• Atoms have a temporary nonsymmetrical distribution of electron density resulting in generation of a temporary dipole
• Temporary dipoles of one molecule induce opposite dipoles in the approaching molecule
Binding Specificity
• Can be absolute or can be very broad• Specificity of racemates may involve E•S complex
formation with only one enantiomer or E•S complex formation with both enantiomers, but only one is converted to product
• Enzymes accomplish this because they are chiral molecules (mammalian enzymes consist of only L-amino acids)
Binding specificity of enantiomers
Scheme 1.2
EnzL + (R,S) EnzL + EnzLR Sdiastereomers
Resolution of a racemic mixture
• Binding energy for E•S complex formation with one enantiomer may be much higher than that with the other enantiomer
• Both E•S complexes may form, but only one E•S complex may lead to product formation
• Enantiomer that does not turn over is said to undergo nonproductive binding
Steric hindrance to binding of enantiomers
Figure 1.4
OOCNH
3
H
OOC NH3
H
A B
S R
Leu
Basis for enantioselectivity in enzymes
Reaction Specificity
Unlike reactions in solution, enzymes can show specificity for chemically identical protons
Figure 1.5
R R'
R R'
Ha
Hb
B
-
enzyme
Enzyme specificity for chemically identical protons. R and R on the enzyme are
groups that interact specifically with R and R, respectively, on the substrate.
Rate Acceleration
• An enzyme has numerous opportunities to invoke catalysis:– Stabilization of the transition state– Destabilization of the E•S complex– Destabilization of intermediates
• Because of these opportunities, multiple steps may be involved
Figure 1.6 1010-1014 fold typically
Catalyzed
Uncatalyzed
Reaction Coordinate
Free Energy (∆G)
A
Uncatalyzed
Enzyme Catalyzed
Reaction Coordinate
Free Energy (∆G)
B
E+S
E+P
ESEP
Effect of (A) a chemical catalyst and (B) an enzyme on activation energy
Enzyme catalysis does not alter the equilibrium of a reversible reaction; it accelerates attainment of the equilibrium
Table 1.2. Examples of Enzymatic Rate Acceleration
Enzyme Nonenzymatic rate knon (s-1)
Enzymatic rate kcat (s-1)
Rate acceleration kcat/knon
cyclophilina 2.8 x 10-2 1.3 x 104 4.6 x 105
carbonic anhydrasea 1.3 x 10-1 106 7.7 x 106
chorismate mutasea 2.6 x 10-5 50 1.9 x 106
chymotrypsinb 4 x 10-9 4 x 10-2 107
triosephosphateisomeraseb
6 x 10-7 2 x 103 3 x 109
fumaraseb 2 x 10-8 2 x 103 1011
ketosteroid isomerasea 1.7 x 10-7 6.6 x 104 3.9 x 1011
carboxypeptidase Aa 3 x 10-9 578 1.9 x 1011
adenosine deaminasea 1.8 x 1010 370 2.1 x 1012
ureaseb 3 x 10-10 3 x 104 1014
alkaline phosphataseb 10-15 102 1017
orotidine 5'-phosphatedecarboxylasea
2.8 x 10-16 39 1.4 x 1017
a Taken from Radzicka, A.; Wolfenden, R. Science 1995, 267, 90.b Taken from Horton, H.R.; Moran, L.A.; Ochs, R.S.; Rawn, J.D.; Scrimgeour,K.G. Principles of Biochemistry; Neil Patterson: Englewood Cliffs, NJ, 1993.
Mechanisms of Enzyme Catalysis
Approximation
• Rate enhancement by proximity• Enzyme serves as a template to bind the
substrates• Reaction of enzyme-bound substrates
becomes first order• Equivalent to increasing the concentration of
the reacting groups• Exemplified with nonenzymatic model studies
Scheme 1.3
CH3COAr
O O
CO
C
O
H3C+ CH3COO-
CH3+ ArO-
Second-order reaction of acetate with aryl acetate
OAr
O
O
O
-
OAr
O
O
O
-
OAr
O
O
O
-
OAr
O
O
O
O
O
-
Relative rate ( krel
)
1 M
-1
s
-1
220 s
-1
5.1 x 10
4
s
-1
2.3 x 10
6
s
-1
1.2 x 10
7
s
-1
Decreasing rotational and
translational entropy
+ CH3
COO
-
OAr
Effective Molarity (EM)
5.1 x 10
4
M
2.3 x 10
6
M
1.2 x 10
7
M
220 M
Table 1.3. Effect of Approximation on Reaction Rates
Covalent Catalysis
Scheme 1.4anchimeric assistance
Most commonCys (SH)Ser (OH)His (imidazole)Lys (NH2)Asp/Glu (COO-)
R Y
O
X X
R Y
O-OR
X
X ZR
O
Activated carbonyl
Z-
+
1.1
-Y-
Nucleophilic catalysis
X-
Scheme 1.5
SCl
SOH
S+
1.2
HO--Cl-
Anchimeric assistance by a neighboring group
Model Reaction for Covalent Catalysis
Scheme 1.6
Early evidence to support covalent catalysis
O
O
18O
O 18OCH3C
18O
18OH
O
OH
18O18
+Ar
H2O
H2O O-
(-ArO-)
General Acid/Base Catalysis
This is important for any reaction in which proton transfer occurs
Figure 1.7catalytic triad
The catalytic triad of a-chymotrypsin. The distances are as follows: d1 = 2.82 Å; d2 =
2.61 Å; d3 = 2.76 Å.
Scheme 1.7
HN
NH
NHR'
Ser OH
R1
O R2
O
R
N N H
His
-OOC Asp
Charge relay system for activation of an active-site serine residue in a-chymotrypsin
• pKa values of amino acid side-chain groups within the active site of enzymes can be quite different from those in solution
• Partly result of low polarity inside of proteinsMolecular dynamics simulations show interiors of these proteins have dielectric constants of about 2-3 (dielectric constant
for benzene or dioxane)• If a carboxylic acid is in a nonpolar region, pKa will
rise• Glutamate-35 in the lysozyme-glycolchitin complex
has a pKa of 8.2; pKa in solution is 4.5• If the carboxylate ion forms salt bridge, it is
stabilized and has a lower pKa
• Basic group in a nonpolar environment has a lower pKa
• pKa of a base will fall if adjacent to other bases
• Active-site lysine in acetoacetate decarboxylase has a pKa of 5.9 (pKa in solution is 10.5)
Two kinds of acid/base catalysis:
• Specific acid or specific base catalysis - catalysis by a hydronium (H3O+) or hydroxide (HO-) ion, and is determined only by the pH
• General acid/base catalysis - reaction rate increases with increasing buffer concentration at a constant pH and ionic strength
Figure 1.8Specific acid/base catalysis General acid/base catalysis
k k
[Buffer] [Buffer]
pH 7.9
pH 7.3
pH 7.9
pH 7.3
A B
Effect of the buffer concentration on (A) specific acid/base catalysis and (B)
general acid/base catalysis
Scheme 1.8
Specific Acid-Base Catalysis
O
C OEt
poornucleophileweak
electrophile
++H3C EtOHCH3COOHH2O
Hydrolysis of ethyl acetate
Scheme 1.9
Alkaline hydrolysis of ethyl acetate
O
COHH3C
O
COC2H5H3C
O
CO-H3C
+ +
strongnucleophile
C2H5O-
HO-
C2H5OH
Scheme 1.10
OC
OHH3C
OHC
OC2H5H3C
OHC
OC2H5H3C
OC
OC2H5H3C +
+
++
strongelectrophile
H3O+ H2OC2H5OH
Acid hydrolysis of ethyl acetate
Scheme 1.11
B+
H
R Y
O
H OHB:
Simultaneous acid and base enzyme catalysis
base catalysis
acid catalysis
Enzymes can utilize acid and base catalysis simultaneously
Simultaneous acid/base catalysis is the reason for how enzymes are capable of deprotonating weak carbon acids
Scheme 1.12
Simultaneous acid and base enzyme catalysis in the enolization of mandelic acid
Ph
HaHO OHb
OPh
O-
O
HaHO
Ph
HaHO OHb
OHc
Ph
HaHO OHb
OHcPh
HO
O-
OHb
Ph
HO
OHc
OHb
pKE = 18.6
+
+
pKa ~ 7.4pKa = 6.6
± Hc+ ± Ha
+
pKE = 15.4
pKa = 22.0
± Ha+
pKa ~ -8
± Hc+
pKa = 3.4
± Hb+
1.3 1.4
1.51.6
• Low-barrier hydrogen bonds - short (< 2.5Å), very strong hydrogen bonds
• Stabilization of the enolic intermediate occurs via low-barrier hydrogen bonds
X R
H
O
H
B:
BH
X R
HO
:B
B+H
R
O
R = H, alkyl, SR'
O–
OX
H HM+
BH
O–
OXH
M+
B
O
O
H
M+
BH
B
H
BB:
B:BH
-HX
A
B
Scheme 1.13
low-barrier H-bond
“weak” base
“strong” acid
“strong” base
“weak” acid
low-barrier H-bond
stronger acid needed
One-base mechanism syn-elimination
carboxylic acids
Simultaneous acid and base enzyme catalysis in the 1,4-elimination of b-substituted (A) aldehydes, ketones, thioesters and (B) carboxylic acids
Two-base mechanism anti-elimination
Scheme 1.14
ElcB mechanism - not relevant
X R
H
O
H
X R
O
B+H
R
O
B:
Base catalyzed 1,4-elimination of b-substituted carbonyl compounds via an enolate
intermediate (ElcB mechanism)Needs acid or metal catalysis
Alternative to Low-Barrier Hydrogen Bond
Scheme 1.15
R
H
O
H
R' R
O
R'
H
B: B+H
Electrostatic enzyme catalysis in enolization
Electrostatic Catalysis
Scheme 1.16
oxyanion hole
HN
NH
HN
NH
NH
HN
NH
HN
O
O
O O
OO
O
R"
R'
RR"
R'
RO
++
also could be aH bond or dipole
Electrostatic stabilization of the transition state
Desolvation
• Exposes substrate to lower dielectric constant environment
• Exposes water-bonded charged groups for electrostatic catalysis
• Destabilizes the ground state
The removal of water molecules at the active site on substrate binding
Scheme 1.17
Strain Energy
k1.8
k1.7 = 108
OP
O HO
-OP
O
O- OO-
-OO
PO
O--O
CH3CH3
OP
O-O
-O
CH3
CH3
HO
1.7 1.8
-OH -OH
Alkaline hydrolysis of phosphodiesters
Figure 1.9
Induced Fit Hypothesisputting strain energy into the substrate
Figure 1.10
Energetic Effect of Enzyme Catalysis
Importance of ground state destabilization
H
Lys252
NH2
NH2
O
COO-
NH2
O
COO-
NH
NH-Lys252
COO-
NH2
COO-B:
NH
NH-Lys252
COO-
NH2
COO-
NH
COO- COO-
H
NH2
NH-Lys252
B
B:H
:B
NH
COO- COO-
NH2
HNH
COO- COO-
NH2
+ +
..
+
+
+
..
:
ZnB(Cys)4
Lys252
NH
NH2
O
COO-
ZnB(Cys)4
H :B
Lys252
NH
NH2OH
COO-
ZnB(Cys)4
Lys252
NH
H2N
COO-
NH2
O
COO- Lys252
NH
N
COO-
HH
:B (X)3ZnA
HO
(X)3ZnA
HO
H
(X)3ZnA
HO
(X)3ZnA
HO
(X)3ZnA
HO
strain energyelectrostatic catalysis
approximationcovalent catalysis
base catalysis
strain energyelectrostatic catalysis
base catalysis
base catalysis
acid catalysis
base catalysis
base catalysis
approximation
approximation
(X3)ZnA (X3)ZnA
Mechanisms of Enzyme Catalysis - porphobilinogen synthase