Enzymes

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Enzymes

• Biological Catalysts
• Nomenclature and Classification
• Enzyme-Substrate Interaction
• Effects of pH and Temperature
• Regulation of Enzyme Activity
• Cofactors and Coenzymes
• Vitamins and Coenzymes

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Enzymes Biological catalysts
Permit reactions to go at body conditions
Large proteins
pH 7.4, 37oC
Process millions of molecules every second
Very specific react with only 1 or a few types
of molecules (substrates).
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Effect of enzymes on Eact
For all reactions you must get over the
activation energy hurdle.
Ea
Reactants H2O2
Energy
?H
Products H2O O2
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Effect of enzymes on Eact
Enzyme catalyzed reaction
Enzymes change how reactions proceed. Reducing ac
tivation energy. Makes faster.
Ea
Reactants H2O2
Energy
?H
Products H2O O2
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Enzyme nomenclature

• Name is based on

what with or how it reacts
-ase ending

Examples To react with lactose. lactase To
remove carboxyl from pyruvate. pyruvate
decarboxylase
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Classification of enzymes

• Based on type of reaction
• Oxireductase
• Transferase
• Hydrolase
• Lyase
• Isomerases
• Ligase

catalyze a redox reaction transfer a functional
group catalyze hydrolysis rxns Add or remove to
CC bonds rearrange to form isomers join two
molecules
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The Active Site

• Enzymes are typically HUGE proteins, yet only a
  small part are actually involved in reaction.

The active site has two basic components. catalyt
ic site binding site
Model of trios-p-isomerase
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The Active Site
Catalytic site Where reaction occurs
Binding Site holds substrate in place
Substrate
Enzyme
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SPECIFICITY

• Enzymes are very specific. Each enzyme will
  catalyze only one type of reactions and often
  will only work with a specific substrate.
• Ex. NH2-C-NH2 H20 2NH3
  CO2
• Urease has no effect on other compounds.
• Such absolute specificity is rather rare among
  enzymes.

urease
O
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Enzyme classes

• Absolutely specific
• Only reacts with a single substrate.
• Group specific
• Works with similar molecules with the same
  functional group.
• Linkage specific
• Catalyzes a specific combination of bonds.
• Stereochemically specific
• Only will work with the proper D- or L- form.

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• ISOENZYMES Different enzymes that perform the
  same type of function in different organisms or
  tissues.

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Enzyme-substrate complex

• Step 1 (All of these steps are in equilibrium)
• Enzyme and substrate combine to form complex
• E S ES
• Enzyme Substrate Complex

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Enzyme-product complex

• Step 2
• An enzyme-product complex is formed.
• ES EP

transition state
ES
EP
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Product

• The enzyme and product separate
• EP E P

The product is made
Enzyme is ready for another substrate.
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Lock and Key Theory

• Enzyme is lock and Substrate is the key.
• Substrate structure
• must fit into enzymes structure.

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Induced Fit Theory

• Active site may not fit substrate.
• Site must change in order to form the complex.

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Effect of Temp on Enzymatic Rxns

• Exceeding normal pH and temperature ranges
  always reduces enzyme reaction rates.

Optimum Temp usually 37oC.
Reaction Rate
Temperature
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Effect of pH on Enzymatic Rxns
Most enzymes work best near pH 7.4 not all
though.
Reaction Rate
pH
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Examples of optimum pH

• Optimum
• Enzyme Source pH
• pepsin gastric mucosa 1.5
• sucrase intestine 6.2
• catalase liver 7.3
• arginase beef liver 9.0
• alkaline bone 9.5
• phosphatase

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Effect of substrate concentration
For non-enzyme catalyzed reactions
Rate of reaction (velocity)
Rate increases if concentration of the substrate
increases
Substrate concentration
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Effect of substrate concentration
For Enzyme catalyzed reactions
Rates increase but only to a certain point
Saturation point
Vmax w/ more enzyme
Vmax w/ some enzyme
At Vmax the enzyme is working as fast as it can.
Rate of reaction (velocity)
Rate is limited by the concentration of both the
substrate and enzyme.
Substrate concentration
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Effect of Enzyme concentration
Enzyme Activity
Enzyme Concentration
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Turnover Number

• Turnover Number
• The rate at which an enzyme transforms the
  substrate
• Is measured at optimum pH and temperature.

Example Carbonic Anhydrase H2CO3
H2O CO2
36,000,000 molecules minute
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ENZYME INHIBITION

• Inhibitors interfere with ability of enzyme to
  react properly with its substrate.
• For example
• Medicinal drugs
• inhibit by inactivating an enzyme essential to
  bacterial growth.
• Viruses more difficult to inhibit because they
  use enzyme system of the host cell.
• (An inhibitor of a virus also
• destroys host cells)

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ENZYME INHIBITION

• Two Types of Inhibitors
• Competitive
• Noncompetitive

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COMPETITIVE INHIBITOR
Competes with substrate for the active site.
Enzyme mistakes inhibitor for substrate
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End product inhibition
Enzyme - substrate reactions in equilibrium.

• E S ES ES EP E
  P

Equilibrium shifts left
Inc P
If product builds up, the reaction slows.
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Reversible Competitive inhibition
Enzyme - substrate reactions in equilibrium.
Inhibitor
Substrate
EI
ES
EI I E S ES
EP ? E P
Shifts
Inc I
Inc S
shifts
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Competitive Inhibitors
Sulfa Drugs

• Illnesses caused by invading microorganisms like
  bacterium can be combated using a competitive
  inhibitor called an antimetabolite.
• Folic Acid is a coenzyme in many biosynthetic
  processes like synthesis of amino acids and
  nucleotides.

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Sulfa Drugs

• Folic Acid obtained
• from the diet or
• from microorganisms in the intestinal tract.

Microorganisms make folic acid from PABA.
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• In 1930 it was discovered that sulfanilamide,
  along with sulfapyridine and sulfathiazole, could
  kill many types of harmful bacteria and help cure
  several diseases.
• The bacteria are tricked into using the sulfa
  drugs instead of PABA.
• They make a molecule that also has a folic acid
  type of structure but is not exactly the same.

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• When they try to use this fake folic acid as a
  coenzyme, not only doesnt it work, it is now a
  competitive inhibitor.
• Many of the bacterias amino acids and
  nucleotides cannot be made, and the bacteria die.

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Penicillin War of Enzyme against Enzyme.

• Produced by mold, it prevents growth of bacteria
  by successfully competing for active sites on an
  enzyme that bacteria need for cell wall
  production.
• 1. Bacteria need the enzyme transpeptidase to
  make their cell walls rigid and cross-linked.
• 2. Penicillin takes control of transpeptidase.
• 3. Bacteria cell walls are not cross-linked and
  the contents of the bacteria cells cannot be
  held in.
• 4. Cytoplasm spills out, and the bacteria die.

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• Penicillin is an example of a beta-lactam.
• It has within its structure the beta-lactam ring.
• Over the past 15-20 years a new strain of
  bacteria has developed that can resist
  penicillin. They contain an enzyme
  (penicillinase) that opens the beta-lactam ring
  and renders the penicillin ineffective.

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By changing the R group, science has found a way
to prevent this from happening.
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Non competitive Inhibition

• This type of inhibitor is believed to alter the
  shape of the enzyme and greatly reduce its
  affinity for the substrate.
• 1. It does not compete with the substrate for
  the active site.
• 2. It does not need to resemble the structure
  of the substrate.
• 3. Its effect cannot be reversed by increasing
  the substrate concentration.

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Non competitive Inhibition

• A noncompetitive inhibitor can bind to an enzyme
  in many ways.
• If it binds somewhere on the surface of the
  enzyme, it causes a change in the tertiary
  structure.
• The substrate is inhibited because it cant get
  into the enzyme.

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Regulation of enzyme activity

• Enzymes are often
• regulated by the cell.
• (Unlike other catalysts)

Cells use several methods to control when
how well enzymes work.
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PROENZYMES (ZYMOGENS)

• Enzymes manufactured in inactive form.

Activated when small part of polypeptide chain
removed.
Hormones, Digestive Enz, Blood Clotting Enz
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PROENZYMES (ZYMOGENS)
Enzymes manufactured in inactive form.
In pancreas (inactive) Proinsulin
In blood (active) Insulin
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PROENZYMES (ZYMOGENS)
(inactive) In pancreas
(active) In Intestines
Trypsinogen
Trypsin
Chymotrypsinogen
Trypsin
Chymotrypsin
procarboxypeptidase
Trypsin
Carboxypeptidase
Digestive Enzymes
Proteases Cleave peptides
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PROENZYMES (ZYMOGENS)
(inactive) In pancreas
(active) In Intestines
Trypsinogen
Trypsin
Chymotrypsinogen
Trypsin
Chymotrypsin
procarboxypeptidase
Trypsin
Carboxypeptidase

• Activation in pancreas rather than intestines ?
• pancreas proteins get digested
• pancreatitis (inflammation of pancreas).

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PROENZYMES (ZYMOGENS)
(inactive) In Gastric mucosa
(active) In Stomach
Pepsinogen
Pepsin
Digestive Enzyme
HCl Produced as Food enters stomach

• As pH ? acid
• Proenzyme gets cleaved
• Pepsin gets activated

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Allosterism FeedBack Control.
Product of one enzyme reaction Controls activity
of another

• A B C D

E1
E2
E3

• Each step is catalyzed by a different enzyme.
• Product D may inhibit the activity of enzyme E1
• When D is low, the 3 reactions proceed rapidly.

• As D increases, E1 becomes inhibited and
  eventually stops.
• Therefore, the accumulation of D is a message
  telling enzyme E1 to shut down.
• This is called feed back control.

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• Similar to coenzymes.
• The presence of an effector molecule alters how
  an enzyme will act.

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Allosteric Enzymes

• Application of non competitive inhibition

• Regulates away from active site

Inactive Enzyme
Active Enzyme
Substrate Now fits
Positive Regulator
Active Site Changed
Positive allosterism - activates the
enzyme. Negative allosterism - deactivates the
enzyme.
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Feedback Control
Inactive Enzyme
Negative Regulator
End Product Stops E1
B
C
D
A
Active Allosteric Enzyme
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• In the body, the protein Kinase is in the
  inactive form most of the time.
• It has a regulator attached that affects the
  active site.
• E R
• The regulator is controlled by another molecule
  called cAMP.
• cyclic Adenosine MonoPhosphate)

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• Whenever the Kinase activity is needed, the body
  sends cAMP to activate the protein Kinase from
  its E-R form.
• E R cAMP

R cAMP
E
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• This is how the ion gates in the postsynaptic
  membane are opened and closed.
• When the neurotransmission stops, cAMP is
  destroyed by the enzyme phosphodiesterase.

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Cofactors

• Apoenzyme
• protein portion
• Inactive

Co2
Co 2
Co2
Cofactor Non protein Group need to activate
apoenzyme
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Mineral Cofactors

• Metal Ion Enzyme involved Function
• Cu2 Cytochrome oxidase redox
• Fe2/Fe3 Catalase redox
• Cytochrome oxidase
• Zn2 Alcohol dehydrogenase Used with
  NAD
• Mg2 Glucose-9-phosphatase
  Hydrolyzes
• phosphate esters

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Coenzymes

• Organic molecule that temporarily binds to
  apoenzyme in order for it to work

Non-Protein
Total
Protein
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HOLOENZYMES AND PROSTHETIC GROUPS

• The Apoenzyme plus the cofactor or coenzyme is
  often referred to as the HOLOENZYME.
• A Cofactor that is tightly bound to the apoenzyme
  is referred to as a PROSTHETIC GROUP.

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Vitamins are oftenconverted to coenzymes

• Vitamin Coenzyme made Function
• B1 thiamine pyrophosphate
  decarboxylation
• B2 flavin mononucleotide carries
  hydrogen
• folic acid tetrahydrofolic acid amino
  acid
• metabolism
• biotin biocytin CO2 fixation
• pantothenic Coenzyme A acyl group carrier
• acid

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Enzymes in Medical Diagnosis and Treatment

• Most enzymes are confined within the cells of the
  body.
• However, small amounts can also be found in body
  fluids (blood, urine, cerebrospinal fluid)
• The level of enzyme activity outside the cells
  can be easily monitored.
• Abnormal activity (high or low) of particular
  enzymes in various body fluids signals either the
  onset of certain diseases or their progression.

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EXAMPLES

• Dead heart muscle cells spill their enzyme
  contents into the serum.
• The level of glutamate oxaloacetate transaminase
  (GOT) in the serum rises rapidly after a heart
  attack.
• The levels of GOT as well as lactate
  dehydrogenase and creatine phosphokinase are
  closely monitored in order to diagnose the
  severity of a myocardial infarction.

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Specific enzyme examples

• Lets look at role of some specific enzymes.
• Two good examples are
• Chymotrypsin
• - A proteolytic enzyme (protein-cleaving).
• - Used in digestion of dietary protein in the
• small intestines.
• Acetylcholinesterase
• - Used for hydrolysis of acetylcholine.
• - Needed for operation of nerves.

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Chymotrypsin
This enzyme is a proteolytic enzyme. It cleaves
peptide bonds.
Peptide bond
This enzyme only works on amino acids containing
an aromatic ring. phenylalanine, tyrosine and
tryptophan.
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Acetylcholinesterase andnerve transmission

• This enzyme is needed to transmit a nerve signal
  at a neuromuscular junction.
• Arrival of a nerve signal causes Ca2 levels to
  increase.
• This causes acetylcholine containing vesicles to
  move to end of the nerve cell where it is
  released.
• Acetylcholine then diffuses across synapse to
  pass the signal to the muscle.
• Acetylcholinesterase then destroys the
  acetylcholine to stop the signal.

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Acetylcholinesterase andnerve transmission
Presence of acetylcholine at receptor causes a
flow of sodium and potassium ions. This causes a
muscle contraction.
synaptic cleft
acetylcholine receptor protein
acetylcholinesterase - destroys excess
acetylcholine
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Acetylcholinesterase
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Acetylcholinesterase andnerve transmission

• Without the enzyme, muscles would continue to
  contract causing spasms.
• Acetylcholinesterase inhibitors are used as
  drugs and poisons.
• Organo fluorophosphates
• - bind to the enzyme. Death can occur.
• Succinylcholine
• Acts like acetylcholine and binds to sites on
  the muscle. Used as a muscle relaxant.

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Another example

• Blood Clotting - formation of fibrin.
• Process requires a series of enzymatic steps.
• Many of the enzymes are made in an inactive
  form. This prevents blood from clotting on its
  own.
• Two pathways can be used to start the process.
• Extrinsic - Activated by tissue damage,
  outside the blood vessel.
• Intrinsic - Activated by damage within a
  blood vessel.

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Summary of pathways
Extrinsic pathway Activation VII VII VII
complex
Intrinsic pathway Activation XII
XII XI XI
IX IX VII
complex
Common pathway X X prothrombin
thrombin fibrin
fibrinogen fibrin polymer
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Fibrin
Ribbon model of fibrin.
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Drug interactions

• Drugs can be administered to alter the clotting
  mechanism.
• Example Heparin - an anticoagulant.
• Acts by accelerating the action of the existing
  inhibitor of thrombin - antithrombin III.
• Antithrombin III inhibits activation of the
  clotting factors that have a reactive serine
  residue at their enzymatically active centers.

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thrombin
antithrombin
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Heparin interaction
thrombin
antithrombin
inhibited thrombin
serine
lysine sites
heparin
Addition of heparin makes it easier for trombin
to interact with antithrombin - positive
allosteric effect.
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Defective enzymesand disease
A number of hereditary diseases result from
the absence of an enzyme or a defective
one. Disease Defective enzyme
Albinism tyrosinase Glactosemia glactose
1-phosphate uridyltransferase
Phenylketonuria phenylalanine hydroxylase
(PKU) Tay-Sachs disease hexosaminidase A
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Phenylketonuria (PKU)

• Genetic mutation that results in a defect of the
  enzyme phenylalanine hydroxylase. (carried by 2
  of population)
• Affects about 1 baby per 13,000.
• Feds may require screening at birth.
• Can result in retarded physical and mental
• development if untreated.
• Treatment - restrict
• phenylalanine until age 10
• (until brain is developed).

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Phenylketonuria (PKU)
PKU is one of a family of enzymatic/genetic
disorders related to phenylalanine metabolism.
tyrosine
phenylalanine
PKU
blocked
albinism
melanin
alkaptonuria
blocked
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ALKAPTONURIA AND OCHRONOSIS

• Alkaptonuria is a rare disease in which the body
  does not have enough of an enzyme called
  homogentisic acid oxidase (HGAO)
• homogentisic acid (HGA) is not used and builds up
  in the body
• Some is eliminated in the urine, and the rest is
  deposited in body tissues where it is toxic.
• The result is ochronosis, a blue-black
  discoloration of connective tissue including
  bone, cartilage, and skin caused by deposits of
  ochre-colored pigment.