Enzyme Catalysis

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Enzyme Catalysis

• 3/17/2003

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General Properties of Enzymes

• Increased reaction rates sometimes 106 to 1012
  increase
• Enzymes do not change DG just the reaction
  rates.
• Milder reaction conditions
• Great reaction specificity
• Can be regulated

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Substrate specificity

• The non-covalent bonds and forces are maximized
  to bind substrates with considerable specificity
• Van der Waals forces
• electrostatic bonds (ionic interactions)
• Hydrogen bonding
• Hydrophobic interaction
• A B P Q
• Substrates Products

enz
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Enzymes are Stereospecific
O
Yeast Alcohol dehydrogenase
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NAD
CH3CD2OH
Ox.
O

CH3C-D
Red.
NADD
Pro-R hydrogen gets pulled off
Yeast Alcohol dehydrogenase
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O
2. NADD CH3-C-H
O
3. CH3-C-D NADH
If the other enantiomer is used, the D is not
transferred
YADH is stereospecific for Pro-R abstraction
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Both the Re and Si faced transfers yield
identical products. However, most reactions that
have an Keq for reduction gt10-12 use the pro-R
hydrogen while those reactions with a Keq lt10-10
use the pro-S hydrogen. The reasons for this are
still unclear
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Specific residues help maintain stereospecificity
Liver alcohol dehydrogenase makes a mistake 1 in
7 billion turnovers. Mutating Leu 182 to Ala
increases the mistake rate to 1 in 850,000. This
is a 8000 fold increase in the mistake rate,
This suggests that the stereospecificity is
helped by amino acid side chains.
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Geometric specificity
Selective about identities of chemical groups
but Enzymes are generally not molecule
specific There is a small range of related
compounds that will undergo binding or catalysis.
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Similar shaped molecules can be highly toxic
Tobacco Nicotine
Because of this closeness in name Nicotinic acid
was renamed to niacin by the bread manufactures
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Coenzymes
Coenzymes smaller molecules that aid in enzyme
chemistry. Enzymes can a. Carry out acid-base
reactions b. Transient covalent bonds c.
Charge-charge interactions Enzymes can not
do d. Oxidation -Reduction reactions e. Carbon
group transfers Prosthetic group - permanently
associated with an enzyme or transiently
associated. Holoenzyme catalytically active
enzyme with cofactor. Apoenzyme Enzyme without
its cofactor
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Commom Coenzymes Coenzyme Reaction
mediated Biotin Carboxylation Cobalamin
(B12) Alkylation transfers Coenzyme A Acyl
transfers Flavin Oxidation-Reduction
Lipoic acid Acyl transfers Nicotinamide
Oxidation-Reduction Pyridoxal Phosphate Amino
group transfers Tetrahydrofolate One-carbon
group transfers Thiamine pyrophosphate Aldehyde
transfer
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Vitamins are Coenzyme precursors
Vitamin Coenzyme Deficiency Disease
Biotin Biocytin not observed Cobalamin
(B12) Cobalamin Pernicious anemia Folic
acid tetrahydrofolate Neural tube
defects Megaloblastic anemia
Nicotinamide Nicotinamide Pellagra Pantothenate
Coenzyme A Not observed Pyridoxine
(B6) Pyridoxal phosphate Not observed Riboflavin
(B2) Flavin Not observed Thiamine
(B1) Thiamine pyrophosphate Beriberi
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These are water soluble vitamins. The Fat
soluble vitamins are vitamins A and D. Humans can
not synthesize these and relay on their presence
in our diets. Those who have an unbalanced diet
may not be receiving a sufficient supply. Niacin
(niacinamide) deficiency leads to pellagra
characterized by diarrhea, dermatitis and
dementia. Pellagra was endemic is Southern
United States in the early 20th century. Niacin
can be synthesized from the essential amino acid,
tryptophan. A corn diet prevalent at the time
restricted the absorption of tryptophan causing a
deficiency. Treatment of corn with base could
release the tryptophan (Mexican Indians treated
corn with Ca(OH)2 before making tortillas!)
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Regulation of Enzymatic Activity
There are two general ways to control enzymatic
activity. 1. Control the amount or availability
of the enzyme. 2. Control or regulate the
enzymes catalytic activity. Each topic can be
subdivided into many different categories.
Enzyme amounts in a cell depend upon the rate in
which it is synthesized and the rate it is
degraded. Synthesis rates can be
transcriptionally or translationally controlled.
Degradation rates of proteins are also
controlled. However, We will be focusing on the
regulation of enzymatic activity.
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The catalytic activity of an enzyme can be
altered either positively (increasing activity)
or negatively (decreasing activity) through
conformational alterations or structural
(covalent) modifications. Examples already
encountered is oxygen, carbon dioxide, or BPG
binding to hemoglobin. Also, substrate binding to
the enzyme may also be modified by small molecule
effectors changing its catalytic site. Protein
phosphorylation of Ser residues can activate or
deactivate enzymes. These are generally
hormonally controlled to ensure a concerted
effect on all tissues and cells.
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Aspartate Transcarbamoylase the first step in
pyrimidine biosynthesis.
ATCase
H2PO4-


Carbamoyl Aspartate
N-Carbamoyl aspartate phosphate
This enzyme is controlled by Allosteric
regulation and Feedback inhibition
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Notice the S shaped curve (pink) cooperative
binding of aspartate Positively homotropic
cooperative binding Hetertropically inhibited by
CTP Hetertropically activated by ATP
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Feedback inhibition Where the product of a
metabolic pathway inhibits is own synthesis at
the beginning or first committed step in the
pathway
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CTP is the product of this pathway and it is also
a precursor for the synthesis of DNA and RNA
(nucleic acids). The rapid synthesis of DNA
and/or RNA depletes the CTP pool in the cell,
causing CTP to be released from ATCase and
increasing its activity. When the activity of
ATCase is greater than the need for CTP, CTP
concentrations rise rapidly and rebinds to the
enzyme to inhibit the activity. ATP activates
ATCase. Purines and Pyrimidines are needed in
equal amounts. When ATP concentrations are
greater than CTP, ATP binds to ATCase activating
the enzyme until the levels of ATP and CTP are
about the same.
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Enzymatic catalysis and mechanisms

• A. Acid - Base catalysis
• B. Covalent catalysis
• C. Metal ion aided catalysis
• D. Electrostatic interactions
• E. Orientation and Proximity effects
• F. Transition state binding
• General Acid Base
• Rate increase by partial proton abstraction by a
  Bronsted base or
• Rate increase by partial proton donation by a
  Bronsted Acid

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Mutarotation of glucose by acid and base catalysts
The reaction can be followed by observation of
the optical activity change
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Kobs apparent first order kinetics but
increases with increased concentrations of acid
and base.
The acid HA donates a proton to ring oxygen,
while the base abstracts a proton from the OH on
carbon 1. To form the linear form. The cycle
reverses itself after attacking the carbonyl from
the other side.
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This compound does not undergo mutarotation in
aprotic solvents. Aprotic solvents have no acid
or base groups i.e. Dimethyl sulfoxide or
dimethyl formamide. Yet the reaction is
catalyzed by phenol, a weak acid and pyridine, a
weak base
v kphenolpyridineTM-a-D-glucose
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The reaction can be catalyzed by the addition of
a-Pyridone as follows
vk'a-pyridoneTM-a-D-glucose k' 7000M x k
or 1M a-pyridone equals phenol70M and
pyridine100M
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Many biochemical reactions require acid base
catalysis

• Hydrolysis of peptides
• Reactions with Phosphate groups
• Tautomerizations
• Additions to carboxyl groups
• Asp, Glu, His, Cys, Tyr, and Lys have pKs near
  physiological pH and can assist in general
  acid-base catalysis.
• Enzymes arrange several catalytic groups about
  the substrate to make a concerted catalysis a
  common mechanism.

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RNase uses a acid base mechanism
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Two histidine residues catalyze the reaction.
Residue His 12 is deprotonated and acts as a
general base by abstracting a proton from the 2'
OH. His 119 is protonated and acts as a general
acid catalysis by donating a proton to the
phosphate group. The second step of the
catalysis His 12 reprotonates the 2'OH and His
119 reacts with water to abstract a proton and
the resulting OH- is added to the phosphate. This
mechanism results in the hydrolysis of the RNA
phosphate linkage.
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Covalent catalysis
Covalent catalysis involves the formation of a
transient covalent bond between the catalyst and
the substrate
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Catalysis has both an nucleophilic and an
electrophilic stage 1 Nucleophilic reaction forms
the covalent bond 2 Withdrawal of electrons by
the now electrophilic catalyst 3 Elimination of
the catalyst (almost the reverse of step 1)
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Depending on the rate limiting step a covalent
catalytic reaction can be either elecrophilic or
nucleophilic. Decarboxylation by primary amines
are electrophilic because the nucleophilic step
of Schiff base formation is very
fast. Nucleophilicity is related to the basicity
but instead of abstracting a proton it attacks
and forms a covalent bond. Lysines are common in
formation of schiff bases while thiols and
imidazoles acids and hydroxyls also have
properties that make good covalent catalysts
Thiamine pyrophosphate and pyridoxal phosphate
also show covalent catalysis
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Metal ion catalysts
One-third of all known enzymes needs metal ions
to work!! 1. Metalloenzymes contain tightly
bound metal ions I.e. Fe, Fe, Cu, Zn,
Mn, or Co. 2. Metal-activated enzymes-
loosely bind ions Na, K, Mg, or Ca. They
participate in one of three ways a. They bind
substrates to orient then for catalysis b.
Through redox reactions gain or loss of
electrons. c. electrostatic stabilization or
negative charge shielding
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Charge stabilization by metal ions
Metal ions are effective catalysts because unlike
protons the can be present at higher
concentrations at neutral pH and have charges
greater than 1.
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Metal ions can ionize water at higher
concentrations
The charge on a metal ion makes a bound water
more acidic than free H2O and is a source of HO-
ions even below pH 7.0
The resultant metal bound OH- is a potent
nucleophile
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Carbonic Anhydrase
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Charge shielding
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Proximity and orientation effects
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k'1 0.0018s-1 when imidazole 1M
When the phenyl acetate form is used k2 0.043
or 24k'1
Proximity effects lead to relatively small rate
enhancement!
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• Reactants are about the same size as water
  molecules (approximation)
• Each species has 12 nearest neighbors (packed
  spheres)
• Reactions only occur between molecules in contact
• Reactant conc. Is low so only one can be in
  contact at a time

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Only a 4.6 rate enhancement but molecular motions
if slowed down leads to a decrease in entropy and
rate enhancements. Molecules are not as reactive
in all directions and many require proper
orientation to react. Increases in rates of 100
fold can be achieved by holding the molecules in
their proper orientation for reaction.
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Preferential transition state binding
Binding to the transition state with greater
affinity to either the product or reactants. RACK
MECHANISM Strain promotes faster rates The
strained reaction more closely resembles the
transition state and interactions that
preferentially bind to the transition state will
have faster rates
kN for uncatalyzed reaction and kE for catalyzed
reaction
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Preferential transition state binding The more
tightly an enzyme binds its reactions transition
state (KT) relative to the substrate (KR) , the
greater the rate of the catalyzed reaction (kE)
relative to the uncatalyzed reaction (kN)
Catalysis results from the preferred binding
and therefore the stabilization of the transition
state (S ) relative to that of the substrate (S).
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106 rate enhancement requires a 106 higher
affinity which is 34.2 kJ/mol
The enzyme binding of a transition state (ES )
by two hydrogen bonds that cannot form in the
Michaelis Complex (ES) should result in a rate
enhancement of 106 based on this effect alone
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Transition state analogues are competitive
inhibitors