Download Chem 306 Ch 19 Enzymes Spring 2007

Ch. 19 - Enzymes and Vitamins
• Living systems are shaped by an enormous variety of
biochemical reactions nearly all of which are mediated by a
series of remarkable biological catalysts known as
enzymes.
• Enzymology (study of enzymes) and biochemistry evolved
together from the 19th century investigation of fermentation.
• “enzyme” – Greek: en, in + zyme, yeast
• E. Buchner showed that alcohol fermentation (ethanol
production from glucose) could be carried out using cell-free
yeast extract.
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What are enzymes?
• Enzymes are biological catalysts.
• Would you expect enzymes to be fibrous or globular proteins?
• They are extremely effective, increasing reaction rates from
106 to 1012 times.
• Most enzymes act specifically with only one reactant (called a
substrate) to produce products
2
• Enzymes facillitate chemical reactions in an active site, a pocket in
an enzyme with the specific shape and chemical makeup necessary
to bind a substrate and where the reaction takes place. The amino
acids His, Cys, Asp, Arg, and Glu participate in 65% of all active sites.
•A living cell has a set of some 3,000 enzymes that it is genetically
programmed to produce. If even one enzyme is missing or defective,
the results can be disastrous.
•Enzymes are used in household products including meat tenderizer,
facial treatments, laundry products, and contact lens cleaners.
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• An example of an enzyme found in many living organisms is
catalase
2 H2O2 (l)  2 H2O (l) + O2(g)
Each molecule of catalase is a tetramer of
four polypeptide chains. Each chain is
composed of more than 500 amino acids.
Located within this tetramer are four
porphyrin heme groups much like the
familiar hemoglobins, cytochromes,
chlorophylls and nitrogen-fixing enzymes in
legumes. The heme group is responsible
for catalase’s enzymatic activity.
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What are some general characteristics of an
enzyme?
• Specificity
Enzymes have a great degree of specificity with respect to
the identities of the substrate (reactants) and their products
than do chemical catalysts.
Absolute specificity:
Enzymes catalyzing of the reaction of one and only
one substance
Relative specificity:
Enzymes that catalyze the reaction of several
structurally related substances
Stereochemical specificity:
Enzymes that are able to distinguish between
stereoisomers
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The specificity of an enzyme for
one of two enantiomers is a
matter of fit. One enantiomer fits
better into the active site of the
enzyme
than
the
other
enantiomer. An enzyme catalyzes
reaction of the enantiomer that fits
better into the active site of the
enzyme.
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Muscle Relaxants and Enzyme Specificity
The mode of action of succinylcholine, a muscle relaxant used
during minor surgery, utilizes the substrate specificity of the
enzyme, acetylcholinesterase.
acetylcholineesterase enzyme
O
O
N+
+ H2O
N+
O
OH
+
OH
Acetylcholine
O
N+
O
O
N+
O
Succinylcholine
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• Catalytic Efficiency
Rates of enzymatically catalyzed reactions are
typically 106 to 1012 times greater than those of the
corresponding uncatalyzed reactions
Turnover number: The number of molecules of
substrate acted on by one molecule of enzyme per
minute
Ex: Carbonic anhydrase converts carbon dioxide to
bicarbonate at a rate of 36 million molecules per
minute.
CO2 + H2O
HCO3- + H+
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• Milder Reaction Conditions
Reactions occur under relatively mild
conditions
Temperature below 100°C,
atmospheric pressure, and nearly
neutral pH
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• Cofactors
Many enzymes are conjugated proteins that require non-protein portions
known as cofactors.
Some cofactors are metal ions.
Carboxypetptidase is an
enzyme that requires a Zn 2+
ion as a cofactor. This enzyme
hydrolyzes the first amide bond
at the C-terminal end of
peptides. Carboxypeptidase is
synthesized in the pancreas
and secreted in the small
intestine.
A “space-filled” representation of
carboxypeptidase
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A proposed model of the active-site chemistry of carboxypeptidase
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Others cofactors are non-protein organic molecules called
coenzymes.
Lactate dehydrogenase is an enzyme that requires the
coenzyme NAD+ /NADH for enzymatic function.
CO2HO
H
CH3
+
NAD+
Lactate
dehydrogenase
CO2C
O
+
NADH +
H+
CH3
Which species have been reduced? Oxidized?
12
Remember, enzymes catalyze both the forward and the
reverse processes.
Biochemical reactions are often represented with the
coenzymes/ enzymes written in conjuction with the equation
“reacts to form” arrow.
13
Structure of NAD+ and NADH
H
H
O
H
O
NH 2
NH 2
-O
P
-O
O
O
O
H
H
H
H
H
OH
H
H
H
OH
NH 2
H
O
O
N
N
P
P
O
H
-O
N
O
N
O
N
N
N
P
O
H
O
H
H
H
OH
H
NAD+
Nicotinamide adenine dinucleotide
N
O
O
O
N
N
-O
O
NH 2
H
H
OH
H
H
H
NADH
Nicotinamide adenine dinucleotide
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• Capacity for Regulation
• Cells control the rates of reactions and the amount
of any given product formed by regulating the
action of the enzyme.
Mechanisms for regulatory process:
Allosteric and feedback control
Covalent modification of the enzyme
Variation of enzyme concentration
15
How are enzymes classified?
Enzymes may be classified according to the type of reaction that they catalyze.
Six main classes:
1. Oxidoreductase: redox reactions
2. Transferase: transfer functional groups
3. Hydrolase: hydrolysis reactions
4. Lyase: addition and elimination reactions
5. Isomerase: isomerization reaction
6. Ligase: bond formation coupled with ATP
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Oxidoreductases
• Catalyze oxidation-reduction (redox) reactions, most
commonly addition or removal of oxygen or hydrogen.
• Requires coenzyme
CO2HO
H
CH3
+
NAD+
Lactate
dehydrogenase
CO2C
O
+
NADH +
H+
CH3
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Transferases
• Catalyze transfer of a functional group from one
molecule to another
• Kinase applied to enzymes that catalyze transfer of
terminal phosphate group
O
HO
-O
CH2
P
O
CH2
O
O
H H
OHOH
H
H
O-
hexokinase
H OH
H H
OHOH
Mg2+
H
OH
ATP
H
H OH
OH
ADP
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Hydrolases
• Catalyze the hydrolysis of substrate – the
breaking of bond with addition of water.
• These reactions are important in the digestive
process.
O
H2C
O
C
(CH2)nCH3
H2C
OH
HC
OH
O
HC
O
C
(CH2)nCH3
+ 3 H2O
Lipase
O
H2C
O
C
+
3 CH3(CH2)nCO2H
(CH2)nCH3
H2C
OH
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Isomerases
• Catalyze the isomerization of a substrate in reactions
that have one substrate and one product
• Rearranges of the functional groups within a molecule
(catalyst converts one isomer in to the other)
CO2CO2HO
H
H
phosphoglycerate
mutase
HO
OO
P
H
O-
O
H
H
O
OH
-O
P
O-
O
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Lyases
• Catalyze the addition of groups such as H2O, CO2, or NH3 to a
double bond or reverse reaction in which a molecule is eliminated to
create a double bond
carbonic
anhydrase
O
C
O
+
H
OH
OH
O
OH
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Ligases
• Catalyze the bonding of two substrate molecules
• reaction where C-C, C-S, C-O, or C-N bond is made or broken
• Accompanied by ATP-ADP conversion (release of energy drives
reaction)
H3C
+
O
O
C
C
CO2
pyruvate
carboxylase
O-
+
ATP
O
-O
C
+
H2C
ADP
+
O
O
C
C
Pi
O-
+
H+
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Subclasses and Types of Reaction
• Oxidoreductase
– Oxidase Oxidation of a
substrate
– Reductase Reduction of a
substrate
– Dehydrogenase: Introduction
of a double bond (C-C or C-O)
• Transferase
– Transaminase Transfer
amino groups
– Kinase Transfers a
phosphate group
• Hydrolyase
– Lipase Hydrolyzes ester
groups of lipids
– Protease Hydrolyzes amide
bonds of proteins
– Nuclease Hydrolyzes
phosphate esters in nucleic
acids
• Lyase
– Dehydrase Loss of water
from a substrate
– Decarboxylase Loss of
carbon dioxide from a
substrate
• Ligase
– Synthetase Formation of a
new C-C bond from two
substrates
– Carboxylase Formation of
a new C-C bond with
carbon dioxide.
• Isomerase
– Epimerase Isomerization
of a chiral carboncenter
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How are enzymes named?
• Common Names
– Derived from the name of the substrate
• Urease
• Lactase
– Derived from the reactions they catalyze
• Dehydrogenase
• Decarboxylase
– Historical Names
• Have no relationship to either substrate or reaction
• catalase, pepsin
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• Systematic Naming
– Unambiguous (often very long)
– Specifies:
• Substrate (substance acted on)
• Functional group
• Type of reaction catalyzed
– Names end in –ase
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Example
O
enzyme
+
H2N
C
H2O
CO2
+ 2 NH3
NH2
• Systematic Name: Urea amidohydrolase
– Substrate: urease
– Functional group: amide
– Type of reaction: hydrolysis
• Common name: Urease
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Can you name the missing enzymes?
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How do enzymes work?
• General Theory
– Substrate and enzyme molecules come into contact and interact
over only a small region of the enzyme surface (active site)
– The substrate bonds to active site via temporay non-covalent
interactions and covalent interactions (less frequently).
– An enzyme-substrate complex (ES) is formed when a substrate
and enzyme bond.
– The substrate is destabilzed in the ES complex by various noncovalent forces.
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• Two models are proposed to represent the interaction between
substrates and enzymes.
These are:
– Lock-and-key model: The substrate is described as fitting into
the active site as a key fits into a lock.
– Induced-fit-model: The enzyme has a flexible active site that
changes shape to accommodate the substrate and facilitate the
reaction.
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30
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• Mechanisms of Catalysis
The chemistry at the active site of an enzyme is the most
important factor in catalytic effect of an enzyme.
• Proximity Effect – the enzyme positions the reactant(s) for a
reaction
• Orientation Effect – positioning of the reactant(s) in the active
site allows for optimum orientation
• Catalytic Effect – atoms at the active site provide structural
features that facillitate the chemistry of the reaction
• Energy Effect – the activation energy requirements for the
reaction are reduced due to any combination of the
above
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Hydrolysis of a peptide bond by chymotrypsin
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How is enzyme activity measured?
• Experiments that measure enzyme activity are called assays.
• Assays for blood enzymes are performed in medical laboratories
– Some assays determine how fast the characteristic color of a
product forms or the color of a substrate decreases
– Some assays are based on reactions in which protons are
produced or used up. This type of enzyme activity can be
followed by measuring how fast the pH of the reacting mixture
changes with time.
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Diagnostically Useful Assays
• Alanine Transamininase (AST)
– Hepatitis
• Lactate dehydrogenase (LDH)
– Heart attacks, liver damage
• Acid phosphatase
– Prostate cancer
• Creatine Kinase (CK)
– Heart attacks
35
36
What are some factors that effect the rate of enzyme
catalyzed reactions?
• Enzyme Concentration
• Substrate Concentration
• Temperature
• pH
37
Effect of Concentration
• Substrate concentration: At low substrate concentration, the reaction
rate is directly proportional to the substrate concentration. With
increasing substrate concentration, the rate drops off as more of the
active sites are occupied.
•Enzyme concentration: The reaction rate varies directly with the
enzyme concentration as long as the substrate concentration does not
become a limitation.
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Change of reaction rate with substrate concentration when enzyme
concentration is constant.
39
Change of reaction rate with enzyme concentration with
no limit on the substrate concentration.
40
Effect of Temperature and pH
• Effect of Temperature: Increase in temperature
increases the rate of enzyme catalyzed reactions. The
rates reach a maximum and then begins to decrease.
The decrease in rate at higher temperature is due to
denaturation of enzymes.
41
Effect of temperature on reaction rate
42
• Effect of pH: The catalytic activity of enzymes depends on pH and
usually has a well defined optimum point for maximum catalytic
activity.
43
How is enzyme activity regulated?
We will see that there are several modes of enzyme
regulation:
• Feedback
• Allosterism
• Inhibition
• Covalent Modification and Genetic Control
Any process that starts or increase the activity of an
enzyme is activation.
Any process that stops or slows the activity of an
enzyme is inhibition.
44
Two of the mechanisms that control the enzymes activity are:
• Feedback control: Regulation of an enzyme’s
activity by the product of a reaction later in a pathway.
45
Example of Feedback Control: Synthesis of Isoleucine
• Threonine deaminase (enzyme that catalyzes 1st step) is subject to
inhibition by the final product (isoleucine)
• Isoleucine binds to a different site on the enzyme and changes the
conformation and threonine binds poorly
• As the concentration of isoleucine increases, the enzyme activity
drops.
H
H
+H N
3
H
C
C
COO-
threonine
deaminase
+H N
3
C
COO-
H
C
CH3
OH
CH2
CH3
threonine
CH3
isoleucine
46
• Allosteric control: Activity of an enzyme is
controlled by the binding of an activator or inhibitor
at a location other than the active site.
Positive allosteric regulator - changes
the active site making the enzyme
a better catalyst (rate accelerates).
Negative allosteric regulator - changes
the active site so that the enzyme becomes
a less effective catalyst (rate decreases).
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A positive regulator
changes the activity site so
that the enzyme becomes
a better catalyst and rate
accelerates.
A negative regulator
changes the activity site
so that the enzyme
becomes less effective
catalyst and rate slows
down.
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• Covalent modification
Enzyme availability can be controlled by storing the enzyme in its
inactive form called zymogens or proenzymes. When the active
enzyme is needed, the stored zymogen is released from storage
and activated at the location of the reaction.
Activation of a zymogen requires a covalent modication of its
structure.
Examples:
– Pepsinogen  pepsin
• Digestion of proteins
– Prothrombin  thrombin
• Blood clotting
49
50
A 3D rendition of the
protease pepsin and its
zymogen form pepsinogen.
Pepsin
Pepsinogen has 44 extra
amino acids (shown in
green) which are removed
when the enzymatic activity
of pepsin is needed.
Pepsinogen
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• Inhibition
The inhibition of an enzyme can be reversible or irreversible.
In reversible inhibition, the inhibitor can leave, restoring the
enzyme to its uninhibited level of activity.
In irreversible inhibition, the inhibitor remains permanently
bound to the enzyme and the enzyme is permanently
inhibited.
Inhibitions are further classified as competitive or noncompetitive.
Non- competitive inhibition if the inhibitor does not compete
with a substrate for the active site.
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Competitive inhibition - the inhibitor competes with a substrate for the
active site.
53
Noncompetitive inhibition - the inhibitor binds elsewhere and not to
the active site.
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•
Genetic control
The supply and synthesis of enzymes is regulated by genes.
Enzymes required at different stages of development are under
genetic control.
Mechanisms controlled by hormones can accelerates or
decelerates enzyme synthesis.
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What are vitamins?
• Vitamins are organic molecules that function in a wide variety of
capacities within the body.
• The most prominent function is as cofactors for enzymatic reactions.
• The distinguishing feature of the vitamins is that they generally
cannot be synthesized by mammalian cells and, therefore, must be
supplied in the diet.
• Vitamins can be classified as water soluble or fat soluble
• Several vitamins work as antioxidants to protect biomolecules from
damage by free radicals.
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Water Soluble Vitamins
•
•
•
•
•
•
•
•
•
Thiamin (B1)
Riboflavin (B2)
Niacin (B3)
Pantothenic Acid (B5)
Pyridoxal,
Pyridoxamine,
Pyridoxine (B6)
Biotin
Cobalamin (B12)
Folic Acid
Ascorbic Acid
.
Fat Soluble Vitamins
•
•
•
•
Vitamin A
Vitamin D
Vitamin E
Vitamin K
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Niacin
Nicotinamide
Nicotinic Acid
• Niacin (nicotinic acid and
nicotinamide) is also known as
vitamin B3
• Niacin is required for the
synthesis of the active forms of
vitamin B3, nicotinamide
adenine dinucleotide (NAD+)
and nicotinamide adenine
dinucleotide phosphate
(NADP+).
• Both NAD+ and NADP+
function as cofactors for
numerous dehydrogenase
enzymes
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