Enzymes

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In the Name of Allah
ENZYMES
TUMS
Make Life on Earth Possible Abolfazl Golestani,
PhD
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An Important Question

• Why should we as medical students, study and
  learn about the ENZYMS?
• For answer go to slide No. 55

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Chemical reaction
Catalyst
A B
Product(s)
Reactant(s)
Catalyst
A B B
C

• Catalysts
• Increase the rate of a reaction
• Are not consumed in the reaction
• Can act repeatedly

Heat Acid Base Metals
What are some of the known catalysts?
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Enzyme is either a pure protein or may require a
non-protein portion

• Apoenzyme protein portion
• Apoenzyme non-protein part Holoenzyme

• According to Holum, the non-protein portion may
  be
• A coenzyme - a non-protein organic substance
  which is loosely attached to the protein part
• A prosthetic group - an organic substance which
  is firmly attached to the protein or apoenzyme
  portion
• A cofactor - these include K, Fe, Fe, Cu,
  Co, Zn, Mn, Mg, Ca, and Mo

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Basic enzyme reactions

• S E ? E P
• S Substrate P Product E Enzyme
• Swedish chemist Savante Arrhenius in 1888
  proposed
• Substrate and enzyme form some intermediate
  known as the Enzyme-Substrate Complex (ES)
• S E ? ES
• ES ? P E

Binding step
Catalytic step
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There are two models of enzyme substrate
interaction
1. Lock and key model Emil Fischer (1890)

• The active site
• Substrate Binding Site
• Catalytic Site

2. Induced fit model Daniel Koshland (1958)
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Induced fit in Carboxypeptidase A
Three amino acids are located near the active
site (Arg 145, Tyr 248, and Glu 270)
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Enzyme-Substrate complex is transient
S E P E

• When the enzyme unites with the substrate, in
  most cases the forces that hold the enzyme and
  substrate are non-covalent.
• Binding forces of substrate are
• Ionic interactions ()(-)
• Hydrophobic interactions (h)(h)
• H-bonds O-H O, N-H O, etc.
• van der Waals interactions

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Some important characteristics of enzymes

• -Potent (high catalytic power) High reaction
  rates
• They increase the rate of reaction by a factor of
  103-1012
• -Efficient (high efficiency)
• catalytic efficiency is represented by Turnover
  number
• ?moles of substrate converted to product per
  second per mole of the active site of the enzyme
  
• -Milder reaction conditions Enzymatically
  catalyzed reactions occur at mild temperature,
  pressure, and nearly neutral pH (i.e.
  physiological conditions)

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Some important characteristics of enzymes, cont.

• -Specific (specificity)
• Substrate specific
• Reaction Specific
• Stereospecific
• -Capacity for regulation
• Enzymes can be activated or inhibited so that
  the rate of product formation responds to the
  needs of the cell
• -Location within the cell
• Many enzymes are located in specific organelles
  within the cell. Such compartmentalization
  serves
• to isolate the reaction substrate from competing
  reactions,
• to provide a favorable environment for the
  reaction, and
• to organize the thousands of enzymes present in
  the cell
• into purposeful pathways.

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Specificity

• Substrate Specificity
• Absolute specificity For example Urease
• Functional Group specificity For example OH,
  CHO, NH2.
• Linkage specificity For example Peptide bond.
• Reaction specificity
• Yields are nearly 100
• Lack of production of by-products
• Save energy and prevents waste of metabolites
• Stereospecificity
• Enzymes can distinguish between enantiomers and
  isomers

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Enzymes requiring metal ions as cofactors
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Many vitamins are coenzyme precursors
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Methods for naming enzymes (nomenclature)

• Very old method Pepsin, Renin, Trypsin
• Old method Protease, Lipase, Urease
• Systematic naming (EC Enzyme Commission
  number)
• The name has two parts
• The first part name of substrate (s)
• The second part ending in ase, indicates
  the type of reaction.
• Additional information can follow in
  parentheses
• L-malateNAD oxidoreductase (decarboxylating)

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Each enzyme has an EC number Enzyme Commission
number

• EC number consists of 4 integers
• The 1st designates to which of the six major
  classes an enzyme belongs
• The 2nd integer indicates a sub class, e.g. type
  of bond
• The 3rd number is a subclassification of the bond
  type or the group transferred in the reaction or
  both (a subsubclass)
• The 4th number is simply a serial number

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There are six functional classes of enzymes
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Enzyme Nomenclature and Classification
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Example of Enzyme Nomenclature

• Common name(s)
• Invertase, sucrase
• Systematic name
• ?-D-fructofuranoside fructohydrolase
• (E.C. 3.2.1.26)
• Recommended name
• ?-fructofuranosidase

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• Kinetic

Enzyme kinetics
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Energy barrier Free Energy of Activation
X T Y
T Transition state
(Ea)
Thermodynamics Type (Exergonic or
Endergonic) Kinetics How fast the reaction will
proceed
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Enzyme Stabilizes Transition State
Whats the difference? Many enzymes function by
lowering the activation energy of reactions.
Adapted from Alberts et al (2002) Molecular
Biology of the Cell (4e) p.166
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EA Activation energy a barrier to the reaction
Can be overcome by adding energy.......
......or by catalysis
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Enzymes Are Complementary to Transition State
X
If enzyme just binds substrate then there will
be no further reaction
Enzyme not only recognizes substrate, but also
induces the formation of transition state, see
also Enz01
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Active Site Is a Deep Buried Pocket
Why energy required to reach transition state is
lower in the active site?
It is a magic pocket
(1) Stabilizes transition

(2) Expels water
CoE
(2)
(1)
(3) Reactive groups
(4)
-
(4) Coenzyme helps
(3)
Juang RH (2004) BCbasics
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Active Site Avoids the Influence of Water

-
Preventing the influence of water sustains the
formation of stable ionic bonds
Adapted from Alberts et al (2002) Molecular
Biology of the Cell (4e) p.115
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Enzyme Reaction Mechanism

• Consider for example the mechanism of
  Chymotrypsin
• Enz06
• Enz07

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Modes of rate enhancement

• Facilitation of Proximity
• Increase the Effective concentration
• Hold reactants near each other in proper
  orientation
• Strain, Molecular Distortion, and Shape Change
• Put a strain on susceptible bonds
• General Acid Base Catalysis
• Transfer of a proton in the transition state
• Covalent Catalysis
• Form covalent bond with substrate
  destabilization of the substrate

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Factors Affecting Rate of Enzyme Reactions

• Temperature
• pH
• Enzyme concentration E
• Substrate concentration S
• Inhibition
• Regulation (Effectors)

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1- Optimum Temperature

• Little activity at low temperature (low number of
  collisions)
• Rate increases with temperature (more successful
  collisions) rate doubles for every 10C
  increase in temperature
• Most active at optimum temperatures (usually 37
  oC in humans)
• Enzymes isolated from thermophilic organisms
  display maxima around 100 C
• Enzymes isolated from psychrophilic organisms
  display maxima around 10 C.
• Activity lost with denaturation at high
  temperatures

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2- Optimum pH

• Effect of pH on ionization of active site
• Effect of pH on enzyme denaturation
• Each enzyme has an optimal pH ( 6 - 8 )
• Exceptions
• digestive enzymes in the stomach (pH
  2)
• digestive enzymes in the intestine
  (pH 8)

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3- Enzyme concentration

• The Rate (v) of reaction Increases proportional
  to the enzyme concentration E (S is high)

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4- Substrate concentration

• When enzyme concentration is constant, increasing
  S increases the rate of reaction, BUT
• Maximum activity reaches when all E combines
  with S (when all the enzyme is in the ES form)

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Enzyme Velocity Curve, see also Enz02
S E
P
(in a fixed period of time)
Constant E
Juang RH (2004) BCbasics
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• Enzymes
• 3rd part

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Michaelis-Menten Equation
S
E
P
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MM Equation Derivation (steady state)
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Practical Summary - Vmax and Km

• Vmax
• How fast the reaction can occur under ideal
  circumstances
• Km
• Range of S at which a reaction will occur
• Binding affinity of enzyme for substrate
• LARGER Km ? the WEAKER the binding affinity

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Practical Summary, cont.
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Practical Summary cont.

• Kcat/Km
• Practical idea of the catalytic efficiency, i.e.
  how often a molecule of substrate that is bound
  reacts to give product

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Order of Reaction

• When S ltlt Km
• vo (Vmax/Km )S
• 2. When S Km
• vo Vmax/2
• 3. When S gtgt Km
• vo Vmax

zero order
Mixed order
2
First order
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Importance of Vi in Measurement of Enzyme
Activity
S
E
P

• Working with vo minimizes complications with
• reverse reactions
• product Inhibition

The rate of the reaction catalyzed by an
enzyme in a sample is expressed in Units. Units
V activity Micromoles (?mol 10-6 mol or
.), of substrate reacting or product produced
per min. It is better to measure it at linear
part of the curve
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Lineweaver-Burk plot
1/2
Km
Direct plot
Double reciprocal plot
Juang RH (2004) BCbasics
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Allosteric Enzymes

• Why the sigmoid shape?
• Allosteric enzymes are multi-subunit enzymes,
  each with an active site
• They show a cooperative response to substrates
• See Enz13

hyperbolic curve Michaelis-Menten kinetics
Sigmoidal curve
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Irreversible InhibitionEnzyme Stops Working
Permanently

• Destruction of enzyme
• Irreversible Inhibitorforms covalent bonds to E
• (inactive E)
• Examples
• Diisopropylfluorophosphate
• inhibits acetylcholine esterase
• binds irreversibly to OH of serine residue
• Cyanide and sulfide
• Inhibit cytochrome oxidase
• bind to the iron atom
• Fluorouracil
• inhibits thymidine synthase (suicide inhibition -
  metabolic product is toxic )
• Aspirin
• Inhibits prostaglandin synthase
• acylates an amino group of the cyclooxygenase

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Reversible InhibitionTemporary Decrease of
Enzyme Function

• Three types based on how increasing S affects
  degree of inhibition
• Competitive degree of inhibition decreases
• Non-competitive degree of inhibition is
  unaffected
• Anti- or Uncompetitive degree of inhibition
  increases
• The Lineweaver-Burk plot is useful in determining
  the mechanisms of actions of various inhibitors,
  see Enz04

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The Effects of Enzyme Inhibitors
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Example

• When a slice of apple is exposed to air, it
  quickly turns brown. This is because the enzyme
• o-diphenyl oxidase catalyzes the oxidation of
  phenols in the apple to dark-colored products.
• Catechol can be used as the substrate. The enzyme
  converts it into o-quinone (A), which is then
  further oxidized to dark products.

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Experiments
No Inhibitor
effect of para-hydroxybenzoic acid (PHBA)
effect of phenylthiourea
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I- Competitive Inhibition
CI
Competitive
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II- Noncompetitive Inhibition
NCI
Noncompetitive (mixed-type)
NCI
S
E
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III- Uncompetitive Inhibition
Uncompetitive (catalytic)
UCI
S
E
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Enzyme Inhibitors in Medicine

• Many current pharmaceuticals are enzyme
  inhibitors (e.g. HIV protease inhibitors for
  treatment of AIDS)
• An example Ethanol is used as a competitive
  inhibitor to treat methanol poisoning
• Methanol
  formaldehyde (very toxic)
• Ethanol competes for the same enzyme
• Administration of ethanol occupies the enzyme
  thereby delaying methanol metabolism long enough
  for clearance through the kidneys

Alcohol dehydrogenase
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Enzymes as diagnostic tools
Enzymes can be used as markers for diagnosis and
prognosis of disease
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Some diagnostically important enzymes
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Useful enzymes for early diagnosis of dental
caries and periodontal disease
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Isozymes of Lactate Dehydrogenase

• Isozymes
• Are catalitically identical (have same catalytic
  activity) BUT physically distinct
• Can be detected by gel electrophoresis (different
  electrical charge)
• Occur in oligomeric enzymes like lactate
  dehydrogenase (LDH)
• In LDH
• Protomers H and M can combine to make five
  different tetramers.

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Isoenzymes of Creatine Kinase

• CK has 3 forms dimer B and M chains
• CK1 BB
• CK2 MB
• CK3MM
• Heart, the only tissue rich in CK2, increases 4-8
  hr after chest pains- peaks at 24 hr.
• LDH peaks 2-3 days after MI.
• New markers
• Troponin T, Troponin I

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Enzymatic control of
Metabolic Pathways
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5- Regulation (Effectors)
Effectors can be classified as follows

• According to type
• Homotropic effector Substrate itself is the
  effector
• Heterotropic effector substance other than
  substrate is the effector
• According to their effect
• Activators (positive effectors)
• Increase the rate of enzyme
• Inhibitors (negative effectors)
• Decrease the velocity of reaction
• Stop the enzyme
• Irreversible
• Reversible
• Competitive
• Non-competitive
• Uncompetitive

Increase or decrease in enzyme reaction rate is
reflected in the graph of V versus S
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Metabolic Pathways

• A metabolic pathway is a chain of enzymatic
  reactions
• Most pathways have many steps, each having a
  different enzyme (E1, E2, E3, E4)
• Step by step, the initial substance used as
  substrate by the first enzyme is transformed into
  a product that will be the substrate for the next
  reaction
• Metabolic regulation is necessary to
• maintain cell components at appropriate levels.
• conserve materials and energy.

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Regulation of Enzyme Activity

• Regulation at transcription level (slowest)
• Isozymes enzymes specific for distinct tissues
  and developmental stages
• Compartmentation of S, E and P
• Specific proteolytic cleavage
• Covalent modification
• (Reversible phosphorylation or adenylation)
• In response to metabolic products (fastest)
• Substrate level control
• Product Inhibition
• Feedback control
• Allosteric Effectors

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Regulation at Transcription Level

• Regulation of E by
• Gene repression
• Induction of genetic expression of enzyme
• There is competition in a cell between the
  processes of protein synthesis and protein
  destruction
• By altering these rates, one can alter the whole
  cell catalytic rate
• It is rather slow

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B. Isoenzymes

• Isozymes provide a means of regulation, specific
  to distinct tissues and developmental stages
• Differential expression of isozymes
• LDH (for example)
• Preferential substrate affinity

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C. Compartmentalization of enzymes

• Substrates and cofactors within the cell are
  also compartmentalized
• Examples
• Enzymes of glycolysis are located in the
  cytoplasm
• Enzymes of citric acid cycle are in the
  mitochondria
• Hydrolytic enzymes are found in the lysosome

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D. Proteolytic activation

• Activation of a zymogen
• Some enzymes are secreted as inactive precursors,
  called zymogens.
• Pancreatic proteases - trypsin, chymotrypsin,
  elastase, carboxypeptidase are all synthesized as
  zymogens trypsinogen, chymotrypsinogen,
  proelastase and procarboypeptidase
• Clotting factors are also part of a proteolytic
  cascade
• Hormone peptides (Pro-insulin Insulin)
• An on/off switch more than regulation

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E. Covalent modification

• Reversible phosphorylation
• Phosphorylation is the most common type of
  modification. Two important classes of enzymes
  are
• Kinases Add a phosphate group to another
  protein/enzyme (phosphorylation)
• transfer of phosphoryl group from ATP to -OH
  group of serine, threonine or tyrosine
• Phosphatases Remove a phosphate group from a
  protein/enzyme (dephosphorylation)

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1- Control of S

• Concentration of substrate and product also
  control the rate of reaction, providing a
  biofeedback mechanism
• Usually 0.1 KmltSPhysiologiclt10 km

Change in enzyme activity
Mild changes in S
Homotropic effectors substrate itself (binding
at different site other than the active site)
affects enzyme activity on other substrate
molecules. Most often this is a positive effector.
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2- Product inhibition

• Enzyme is reversibly inhibited by the product
• Example hexokinase in the first reaction of
  glycolysis is inhibited by glucose-6-phosphate
  (G6P the product)

glucose ATP glucose-6-phosphate
ADP 
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Why? As v approaches Vmax, the product becomes
significant, and can compete with the substrate
for the enzyme. The product becomes a
competitive inhibitor and slows down activity of
the enzyme.
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3- Negative feedback control(end product
inhibition)

• Final product of a metabolic sequence feeds-back
  negatively on early steps
• In feedback inhibition, there is a second binding
  site on the enzyme where the inhibitor binds, so
  that the inhibitor is not necessarily similar in
  structure to the substrate

Enz 2
Enz 1
Enz 3
Enz 4
A B C
D E
_

• What happens?
• As the need for product E decreases, E will
  accumulate
• Most efficient to inhibit at first step of the
  pathway, slow the first reaction so intermediates
  do not build up 
• An increase in the concentration of E, leads to a
  decrease in its rate of production of E

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Regulation of the metabolism, feed-back
inhibition by the final product - end product
inhibition
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4- Positive feedforward control

• Earlier reactants in a metabolic sequence
  feed-forward positively on later steps.

If A is accumulating, it speeds up downstream
reactions to use it up

Metabolism involves the complex integration of
many feedback and feedforward loops
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4- Allosteric control

• Allosteric activator stabilizes active "R" state
• shift the graph to the left
• Allosteric inhibitor stabilizes less active or
  inactive "T" state
• shift the graph to the right

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Multi reactant enzymes
have more than one substrate
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Multi reactant enzymes reactancy

• Published by W. W. Cleland in1963
• Nomenclature is based on number of substrates and
  products in the reaction.
• Reactancy the number of kinetically significant
  substrates or products and designated by
  syllables Uni, Bi, Ter, Quad.

A ? P Uni Uni A ? P Q Uni Bi A B ? P
Q Bi Bi A B C ? P Q R S Ter Quad
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Multi reactant enzymes mechanism

• Sequential - if all S add to E before any P are
  released.
• Sequential ordered - if S add in an obligatory
  order (two on two off)
• Sequential random - if S do not add in
  obligatory order (two on two off)
• Ping Pong - If one or more S released before all
  S bind
• (one on, one off one on, one off)
• Note there is some sort of modified enzyme
  intermediate (often covalent intermediate)

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Random sequential (example)
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Ordered sequential (example)
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Ping pong or double displacement mechanism
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Double displacement (example)
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Other kinds of enzymes

• Some ribonucleoprotein enzymes have been
  discovered
• The catalytic activity is in the RNA part
• They are called Ribozymes
• Catalytic antibodies are called Abzymes

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