Neurotransmitter

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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
  

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• 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
catalases 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.
Acetylcholine
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 100C, 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. Which species have been
reduced? Oxidized?
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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.
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Structure of NAD and NADH
NAD Nicotinamide adenine dinucleotide
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

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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

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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

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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.

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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)

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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

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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)

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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

• 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 non-covalent 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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• 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

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What are some factors that effect the rate of
enzyme catalyzed reactions?

• Enzyme Concentration
• Substrate Concentration
• Temperature
• pH

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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.
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Change of reaction rate with enzyme
concentration with no limit on the substrate
concentration.
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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.

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Effect of temperature on reaction rate
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• Effect of pH The catalytic activity of enzymes
  depends on pH and usually has a well defined
  optimum point for maximum catalytic activity.

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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.

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Two of the mechanisms that control the enzymes
activity are

• Feedback control Regulation of an enzymes
  activity by the product of a reaction later in a
  pathway.

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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.

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• 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

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A 3D rendition of the protease pepsin and its
zymogen form pepsinogen. Pepsinogen has 44
extra amino acids (shown in green) which are
removed when the enzymatic activity of pepsin is
needed.
Pepsin
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 non-competitive.
• 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.

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• 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

• 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