Download Figure 15-1a Mechanisms of keto–enol tautomerization. (a

Enzymatic Catalysis
Instructor: Dr. Tsung-Lin Li
Genomics Research Center
Academia Sinica
Source book: Biochemistry (3rd) by Voet and Voet
Catalytic mechanisms
1.
2.
3.
4.
5.
6.
The specificity of substrate binding combined
with the optimal arrangement of catalytic
groups (as a result of eons of evolution)
Acid-based catalysis
Covalent catalysis
Metal ion catalysis
Electrostatic catalysis
Proximity and orientation effects
Preferential binding of the transition state
complex
Acid-base catalysis
• General acid catalysis is a process in which partial
proton transfer from a Brønsted acid (a species
that can donate protons) lowers the free energy of
a reaction’s transition state.
• General base catalysis is a process if the rate is
increased by partial proton abstraction by a
Brønsted base (a species that can combine with a
proton).
• Some reactions may be simultaneously subject to
both processes: a concerted general acid-base
catalyzed reaction
A Mechanisms of keto–enol tautomerization.
(a) Uncatalyzed. (b) General acid catalyzed. (c) General base catalyzed.
RNase A–catalyzed hydrolysis
The bovine pancreatic RNase A–catalyzed hydrolysis of RNA
is a two-step process with the intermediate formation of a
2,3 -cyclic nucleotide.
RNase A–catalyzed hydrolysis
The pH dependence of Vmax/KM in the RNase A–catalyzed
hydrolysis of cytidine-2,3 -cyclic phosphate.
Covalent catalysis
• Covalent catalysis involves rate acceleration through
the transient formation of a catalyst-substrate bond.
The decarboxylation of acetoacetate.
Covalent catalysis has both nucelophilic and
electrophilic stages
• The nucleophilic reaction between the
catalyst and the substrate to form a covalent
bond
• The withdrawal of electrons from the
reaction center by the now electrophilic
catalyst
• The elimination of the catalyst, a reaction
that is essentially the reverse of stage 1
Certain amino acid side chains and coenzymes
can serve as covalent catalysts
• Residues: Lys, His, Cys, Asp, Ser
• Cofactors: TPP, PLP
Metal ion catalysis
• About 1/3 of all known enzymes require the
presence of metal ions for catalytic catalytic
activity
• Two classes of metal ion-requiring enzymes that
are distinguished by the strengths of their ionprotein interactions
 Metalloenzymes
Contain tightly bound metal ions
(transition metal ions) such as Fe2+, Fe3+,
Cu2+, Zn2+, Mn2+, or Co2+
 Metal-activated enzymes
Loosely bind metal ions from solution,
(usually alkali and alkaline earth metal
ions) such as Na+, K+, Mg2+, or Ca2+
 Metal ions participate the catalytic process
in three major ways
1. By binding to substrates so as to orient
them properly for reaction
2. By mediating oxidation-reductions
through reversible changes in the metal
ion’s oxidation state.
3. By electrostatically stabilizing or shielding
negative charges
Metal ions promote catalysis through
charge stabilization
• The metal ion acts in much the same way as a proton to
neutralize negative charge, that is, it acts as a Lewis acid.
• Metal ions are often much more effective catalysts than
protons because metal ions can be present in high
concentrations at neutral pH’s and can have charges
greater than +1.
• Metal ions have been dubbed “superacids”.
Metal ions promote nucleophilic catalysis
via water ionization
• A metal ion’s charge makes its bound water molecules
more acidic than free H2O and therefore a source of OHions even below neutral pH’s.
• For example,
(NH3)5Co3+(H2O)
(NH3)5Co3+(OH-) + H+
CO2 + H2O
HCO3- + H+
X-Ray structures of human carbonic anhydrase. (a) Its active
site in complex with bicarbonate ion.
X-Ray structures of human carbonic anhydrase. (b) The
active site showing the proton shuttle.
•Through general base catalysis by His64 (but too far away from Zn2+bound water), they are two intervening water molecules to form a
hydrogen bonded network that act as a proton shuttle.
•The resulting Zn2+-bound OH- ion nucleophilically attacks the nearby
enzymatically bound CO2, thereby converting it to HCO3-.
•The Zn2+-bound OH- group donates a hydrogen bond to Thr199, which
in turn donates a hydrogen bond to Glu106. These interactions orient the
OH- group with the optimal geometry for nucleophilic attack on the
substrate CO2.
Metal ions promote reactions through
charge shielding
• The actual substrates of kinases are Mg2+-ATP
complexes rather than just ATP.
Electrostatic catalysis
• Charge distributions about the active sites of
enzymes are arranged so as to stabilize the
transition states of the catalyzed reactions.
• Such a mode of rate enhancement, which
resembles the form of metal ion catalysis, is
termed electrostatic catalysis.
• These charge distributions serve to guide polar
substrates toward their binding sites so that the
rates of these enzymatic reactions are greater than
their diffusion-controlled limits
catalysis through proximity and
orientation effects
• Enzyme catalytic mechanisms are far more
efficient than those of organic model reactions
due to effects of proximity and orientation.
• Reactants must come together with the proper
spatial relationship for a reaction to occur
It is 24-fold more effective
• Proximity along contribute relatively little to
catalysis; factors that will increase this value
other than proximity alone clearly must be
considered.
• Properly orienting reactants and arresting
their relative motions can result in large
catalytic rate enhancements
Reactants react most readily only if they have the proper relative
orientation
The geometry of an SN2 reaction.
Relative Rates of Anhydride Formation for Esters Possessing
Different Degrees of Motional Freedom in the Reaction
Above.
Catalysis by preferential transition state binding
• The binding of the transition state to an enzyme
with greater affinity than the corresponding
substrates or products.
• Rack mechanism: enzymes mechanically strained
their substrates toward the transition state
geometry through binding sites into which
undistorted substrate did not properly fit.
• The strained reactant more closely resembles the
transition state of the reaction than does the
corresponding unstrained reactant.
Reaction coordinate diagrams for a hypothetical enzymatically catalyzed
reaction (single substrate - blue; corresponding uncatalyzed reaction red).
The effect of preferential transition state binding
Transition state analogs are competitive inhibitors
• Transition state analogs, stable molecules that resemble S or
one of its components, are potent competitive inhibitors of
the enzyme.
X
O
Lysozme
The alternating NAG–NAM polysaccharide component of bacterial cell
walls.
Enzyme structure
Primary structure of HEW lysozyme.
X-Ray structure of HEW lysozyme. (c) A computer-generated
model showing the protein’s molecular envelope (purple) and
Ca backbone (blue).
X-Ray structure of HEW lysozyme. (b) A ribbon diagram of
lysozyme highlighting the protein’s secondary structure.
Table 15-2
Rates of HEW Lysozyme-Catalyzed
Hydrolysis of Selected Oligosaccharide Substrate Analogs.
X-Ray structure of HEW lysozyme. (a) The polypeptide chain is shown
with a bound (NAG)6 substrate (green).
The fourth NAG residue (D) appeared unable to bind to the enzyme
because it C6 and O6 atoms too closely contact Glu35, Trp108, and the
acetamido group of NAG residue C.
This steric interference could be relieved by distorting the glucose ring
from its normal chair conformation to that of a half-chair.
Chair and half-chair conformations.
Interactions of lysozyme with its substrate.
•This distortion moves the –C6H2OH group
from its normal equatorial position to an axial
position where it makes no close contacts and
can hydrogen bond to the backbone carbonyl
group of Gln57 and the amido group of Val109
•The lactyl side chain of NAM cannot be
accommodated in the binding subsites of either
residues C or E. Hence, NAM residues must
bind to the enzyme in substies B, D and F.
•The probable cleavage site is between residues
D and E. (Or, between second and third residues
from its reducing terminus).
Catalytic mechanism
Mechanism of the nonenzymatic acid-catalyzed hydrolysis of an acetal
to a hemiacetal.
The Phillips mechanism for the lysozyme reaction.
1. Asp52 is surrounded by
polar residues and thus
predicted to have a normal
pK ( unprotonated)
2. Glu35 is nestled in a
nonpolar pocket, and is thus
likely to remain protonated
(C1, C2, C5, and C5 are coplanar)
The D-ring oxonium ion intermediate in the Phillips
mechanism is stabilized by resonance.
Testing the Phillips mechanism
1. Identification of the catalytic residues (site-directed mutagenesis).
2. Role of strain
The d-lactone analog of (NAG)4.
Binding Free Energies of HEW Lysozyme Subsites.
3. The lysozyme reaction proceeds via a covalent intermediate
For all b-glycosidase but not lysozyme
Probing the adduct by incubating the analogue
with E35Q mutant (ESI/MS)
(X-ray)
The HEW lysozyme covalent intermediate (a double displacement mechanism).
Serine Proteases
Chymotrypsin, trypsin and elastase
A. Kinetics and catalytic groups
a. Ester hydrolysis as a kinetic model
Burst phase (stoichiometric)
Steady state phase (rate is
constant and independent with
substrate conc.
Ping pong Bi Bi
A Selection of Serine Proteases.
b. Identification of the catalytic residues
Chemically labeling
Chymotrypsin Ser195
(a serine esterase)
(insecticides)
Reaction of TPCK with chymotrypsin to alkylate His 57.
Affinity labeling
(a substrate analog bearing a reactive
group)
Chymotrypsin His57
B. Structure
X-Ray structure of bovine trypsin.
(a) A drawing of the enzyme in complex. (b) A ribbon diagram of trypsin.
X-Ray structure of bovine trypsin. (c) A drawing showing the
surface of trypsin (blue) superimposed on its polypeptide
backbone (purple).
The active site residues of chymotrypsin.
The catalytically essential His57, and Ser195 are located at the substrate
binding site together with the invariant Asp102, which is buried in a
solvent-inaccessible pocket. These three residues form a hydrogen
bonded constellation referred to as the catalytic triad.
Serine Protease Catalysis
• Serine proteases are enzymes that catalyze the hydrolysis of peptide bonds. In each
case, the enzymes have a serine residue that plays a critical role in the catalysis. The
enzymes cuts preferentially in distinct sites. The active site regions of all of the
serine proteases have a number of common factors. For example, an aspartate
residue, a histidine residue, and a serine residue are always clustered about the
active site depression.
•The shape and charge of the "pocket," however, vary between different
serine proteases. Thus, it is the nature of the pocket that gives a serine
protease its specificity. For example, in chymotrypsin, the pocket is wide
and lined with hydrophobic residues to accommodate a hydrophobic side
chain, such as phenylalanine.
Substrate specificity
b. Evolutionary relationships among serine proteases
• The sequence and structural homologies
among proteins reveal their evolutionary
relationships.
• The greater similarities among
chymotrypsin, trypsin, and elastase indicate
that these proteins evolved through gene
duplications of an ancestral serine protease
followed by the divergent evolution of the
resulting enzymes.
Relative positions of the active site residues in
subtilisin, chymotrypsin, serine carboxypeptidase II, and ClpP protease.
These proteins apparently constitute a remarkable example of convergent
evolution: nature seems to have independently discovered the same
catalytic mechanism at least four times.
Catalytic mechanism
The catalytic mechanism of chymotrypsin.
These steps include the following:
1. Polypeptide substrate binding.
2. Proton transfer from Ser to His. The substrate forms a tetrahedral transition state with
the enzyme.
3. Proton transfer to the C-terminal fragment, which is released by cleavage of the C-N
bond. The N-terminal peptide is bound through acyl linkage to serine.
4. A water molecule binds to the enzyme in place of the departed polypeptide.
5. The water molecule transfers its proton to His 57. Again, a tetrahedral transition state
is formed.
6. The second peptide fragment is released. The acyl bond is cleaved, the proton is
tranferred from His back to Ser, and the enzyme returns to its initial state.
A key to the mechanism of serine protease catalysis lies in the stability of the two
tetrahedral intermediate states, which are very similar to the essential transition states.
They appear to be stabilized by hydrogen bonds from backbone amino protons from
residues Ser 195 and Gly 193 to one of the oxygens in the tetrahedral complex (the
carbonyl oxygen of the substrate). The hydrogen bonding can occur only with formation
of the tetrahedral state and thus stabilizes the intermediates.
Serine proteases have a histidine and an acidic residue in their active site. Histidine is
very common in active sites, because it readily accepts or donates protons at
physiological pH.
Catalysis of peptide bond hydrolysis by chymotrypsin.
Catalytic mechanism of the serine proteases.
Catalytic mechanism of serine proteases
D. Testing the catalytic mechanism
a. The tetrahedral intermediate is mimicked in a complex of trypsin with
trypsin inhibitor (BPTI)
Trypsin–BPTI complex. (a) The X-ray structure shown as a cutaway
surface drawing indicating how trypsin (red) binds BPTI (green). (b)
Trypsin Ser 195, the active Ser, is in closer-than-van der Waals contact
with the carbonyl carbon of BPTI’s scissile peptide.
b. Serine protease preferentially bind the transition state
The conformational distortion that occurs with the formation of the
tetrahedral intermediate causes the carbonyl oxygen of the scissile
peptide to mover deeper onto the active site so as to occupy a previously
unoccupied position, the oxyanion hole.
Transition state stabilization in the serine proteases. (a) The
Michaelis complex. (b) The tetrahedral intermediate.
c. The tetrahedral intermediate and the water molecule attacking the acylenzyme intermediate have been directly observed.
X-Ray structures of porcine pancreatic elastase in complex
with the heptapeptide BCM7 (YPFVEPI). (a) The complex at
pH 5. (b) The complex at pH 9.
d. The role of the catalytic triad: low-barrier hydrogen bonds.
Implausible?
The hydrogen atom becomes more or less equally shared between donor
and acceptor, when pK’s of both are nearly equal. (D H---A)
In nonaqueous solution or in enzyme active site, LBHB can occur.
e. Much of a serine protease’s catalytic activity arises from preferential
transition state binding.
1010
106
104
Blocking all catalytic triad
As a consequence, a large portion of chymotrypsin’s rate enhancement
must be attributed to its preferential binding of the catalyzed reaction’s
transition state.
Zymogen
Activation of trypsinogen to form trypsin.
Activation of chymotrypsinogen by proteolytic cleavage.
Hypothetical QSAR plots of log(1/C) versus log P for a series
of related compounds. (a) A plot that is best described by a
linear equation.
Hypothetical QSAR plots of log(1/C) versus log P for a series
of related compounds. (b) A plot that is best described by a
quadratic equation.
The combinatorial synthesis of arylidene diamides.
X-Ray structure of cytochrome P450CAM from Pseudomonas
putida showing its active site region.
The metabolic reactions of acetaminophen that convert it to
its conjugate with glutathione.
The assembly, budding, and maturation of HIV-1.
HIV-1 polyproteins. (a) The organization of the HIV-1 gag and
gag–pol polyproteins.
HIV-1 polyproteins. (b) The sequences flanking the HIV-1
protease cleavage sites (red bonds) indicated in Part a.
Renin participation in blood pressure regulation.
X-Ray structure of pepsin. (a) Ribbon diagram.
X-Ray structure of pepsin. (b) Enlarged view of the active site
Asp residues and their bound water molecule indicating the
lengths (in Å) of possible hydrogen bonds (gray).
Catalytic mechanism of aspartic proteases.
X-Ray structure of HIV-1 protease. (a) Uncomplexed.
X-Ray structure of HIV-1 protease. (b) In complex with its
inhibitor.
Arrangement of hydrogen bonds between HIV-1 protease
and a modeled substrate.
Comparison of a normal peptide bond to several groups (red)
that are similar to the tetrahedral intermediate of aspartic
proteases.
Some HIV-1 protease inhibitors that are in
clinical use.
Mechanism of carboxypeptidase A.