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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

Ch106; Chpt 21 Enzymes                                  20 - 1
                         Enzymes = Biological catalysts
                                   Permit reactions
        Large proteins                 to „go‟ at
                                   body conditions
                                           pH 7.4, 37oC
                                    Process millions
                                      of molecules
                                      every second
                                      Very specific
                                    react with only
                                    1 or a few types
                                      of molecules
                                      (substrates).
Ch106; Chpt 21 Enzymes                                    20 - 2
                              Effect of enzymes on Eact

                                        For all reactions
                                        you must get over
                                        the activation
                                        energy hurdle.
                                  Ea
               Reactants
     Energy




                 H2O2

                         H                Products
                                           H2O + O2



Ch106; Chpt 21 Enzymes                                 20 - 3
                              Effect of enzymes on Eact
    Enzyme catalyzed                     Enzymes change
        reaction                          how reactions
                                            proceed.
                                            Reducing
                                  Ea    activation energy.
               Reactants
     Energy




                 H2O2                     Makes faster.

                         H                Products
                                           H2O + O2



Ch106; Chpt 21 Enzymes                                    20 - 4
                                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

Ch106; Chpt 21 Enzymes                           20 - 5
                         Classification of enzymes
    Based on type of reaction
       Oxireductase catalyze a redox reaction
       Transferase        transfer a functional group

       Hydrolase          catalyze hydrolysis rxns

       Lyase              Add or remove to C=C bonds
       Isomerases         rearrange to form isomers

       Ligase             join two molecules

Ch106; Chpt 21 Enzymes                                  20 - 6
                                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.
                                 catalytic site
                                 binding site
                                       Model of
                                  trios-p-isomerase


Ch106; Chpt 21 Enzymes                                20 - 7
                                  The Active Site



                                  Catalytic site
    Binding                        Where reaction
                                      occurs
      Site
        holds
      substrate
          in
        place                           Substrate

                         Enzyme
Ch106; Chpt 21 Enzymes                              20 - 8
                                          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   urease   2NH3 + CO2
                         O

     • Urease has no effect on other compounds.
     • Such absolute specificity is rather rare among
       enzymes.


Ch106; Chpt 21 Enzymes                                20 - 9
                                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.
Ch106; Chpt 21 Enzymes                               20 - 10
   • ISOENZYMES: Different enzymes that perform
     the same type of function in different
     organisms or tissues.




Ch106; Chpt 21 Enzymes                        20 - 11
                             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




                         +



Ch106; Chpt 21 Enzymes                             20 - 12
                          Enzyme-product complex
     Step 2:
     An enzyme-product complex is formed.

              ES                           EP



     ES                  transition   EP
                            state



Ch106; Chpt 21 Enzymes                          20 - 13
                                           Product


    The enzyme and product separate

                     EP       E + P
                                          The product
                                            is made
                             Enzyme is
                               ready
                  EP            for
                              another
                             substrate.


Ch106; Chpt 21 Enzymes                             20 - 14
Ch106; Chpt 21 Enzymes   20 - 15
                         Lock and Key Theory

     Enzyme is “lock” and Substrate is the “key”.
                  Substrate structure
           must fit into enzyme‟s structure.




Ch106; Chpt 21 Enzymes                              20 - 16
                                    Induced Fit Theory
                  Active site may not fit substrate.

     Site must change in order to form the complex.




Ch106; Chpt 21 Enzymes                                 20 - 17
Ch106; Chpt 21 Enzymes   20 - 18
                       Effect of Temp on Enzymatic Rxns
      Exceeding normal pH and temperature ranges
      always reduces enzyme reaction rates.

                                         Optimum Temp
       Reaction Rate




                                            usually
                                             37oC.




                           Temperature

Ch106; Chpt 21 Enzymes                                  20 - 19
                       Effect of pH on Enzymatic Rxns


                                     Most enzymes
                                  work best near pH 7.4
       Reaction Rate




                                     not all though.




                           pH
Ch106; Chpt 21 Enzymes                                20 - 20
                          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


Ch106; Chpt 21 Enzymes                              20 - 21
                       Effect of substrate concentration

        For non-enzyme catalyzed reactions
    Rate of reaction
       (velocity)




                                           Rate increases if
                                            concentration of
                                        the substrate increases

                             Substrate concentration
Ch106; Chpt 21 Enzymes                                            20 - 22
                       Effect of substrate concentration
      For Enzyme catalyzed reactions

                       Rates increase but only to a certain point
                        Vmax w/ more enzyme   Saturation point
    Rate of reaction




                       Vmax w/ some enzyme             At Vmax
       (velocity)




                                              the enzyme is working as
                                                    fast as it can.
                                                  Rate is limited by
                                              the concentration of both
                                             the substrate and enzyme.

                               Substrate concentration
Ch106; Chpt 21 Enzymes                                                    20 - 23
                    Effect of Enzyme concentration



     Enzyme
     Activity




                         Enzyme Concentration



Ch106; Chpt 21 Enzymes                          20 - 24
                              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
Ch106; Chpt 21 Enzymes                          20 - 25
                             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)

Ch106; Chpt 21 Enzymes                                  20 - 26
                           ENZYME INHIBITION

   • Two Types of Inhibitors:
     – Competitive

        – Noncompetitive




Ch106; Chpt 21 Enzymes                    20 - 27
                          COMPETITIVE INHIBITOR



                                   Competes with
                                    substrate for
                                   the active site.


                Enzyme
                mistakes
              inhibitor for
               substrate
Ch106; Chpt 21 Enzymes                                20 - 28
                  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
Ch106; Chpt 21 Enzymes                                     20 - 30
                         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.




Ch106; Chpt 21 Enzymes                              20 - 31
                                                    O
     Sulfa Drugs                                    C
                                                         OH
     Folic Acid : obtained
                                  H N
     • from the diet or                  p-aminobenzoic acid
                                    H
     • from microorganisms in the intestinal tract.
     Microorganisms make folic acid from PABA.
      H2N         N      N
                                     O                     O
              N              NH         C NH CH C OH
                         N
                                                   CH2
                  OH
                                                   CH2
                                                   C OH
                                                   O
Ch106; Chpt 21 Enzymes                                         20 - 32
Ch106; Chpt 21 Enzymes   20 - 34
                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.
Ch106; Chpt 21 Enzymes                              20 - 36
    By changing the R group, science has found a
    way to prevent this from happening.
Ch106; Chpt 21 Enzymes                             20 - 38
                         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.



Ch106; Chpt 21 Enzymes                            20 - 39
                         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 can‟t get into the
   enzyme.




Ch106; Chpt 21 Enzymes                          20 - 40
                         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.




Ch106; Chpt 21 Enzymes                                       20 - 41
                         PROENZYMES (ZYMOGENS)
    Enzymes manufactured in inactive form.
                  Activated when small part
                  of polypeptide chain removed.




  Hormones,
  Digestive Enz,
  Blood Clotting Enz
Ch106; Chpt 21 Enzymes                            20 - 42
                         PROENZYMES (ZYMOGENS)
    Enzymes manufactured in inactive form.
    In pancreas (inactive)     In blood (active)
            Proinsulin             Insulin


              S                    S
                  S      S             S   S
                         S                 S
              S                    S
                  S                    S




Ch106; Chpt 21 Enzymes                             20 - 43
                         PROENZYMES (ZYMOGENS)
               (inactive)                        (active)
              In pancreas                     In Intestines

       Trypsinogen         enteropeptidase    Trypsin


  Chymotrypsinogen            Trypsin     Chymotrypsin

procarboxypeptidase Trypsin Carboxypeptidase

          Digestive           Proteases
          Enzymes           Cleave peptides

Ch106; Chpt 21 Enzymes                                        20 - 44
                         PROENZYMES (ZYMOGENS)
               (inactive)                       (active)
              In pancreas                    In Intestines

       Trypsinogen         enteropeptidase   Trypsin


  Chymotrypsinogen            Trypsin   Chymotrypsin

procarboxypeptidase Trypsin Carboxypeptidase
  Activation in pancreas rather than intestines 
    •pancreas proteins get digested
    •pancreatitis (inflammation of pancreas).
Ch106; Chpt 21 Enzymes                                       20 - 45
                         PROENZYMES (ZYMOGENS)
            (inactive)                        (active)
        In Gastric mucosa                   In Stomach

     Pepsinogen                  H+             Pepsin


          Digestive              HCl
          Enzyme             Produced as
                          Food enters stomach
  As pH  acid
    •Proenzyme gets cleaved
    •Pepsin gets activated
Ch106; Chpt 21 Enzymes                                   20 - 46
                            Allosteric Enzymes
    • Application of non competitive inhibition
    • Regulates away from active site
             Inactive     Active        Substrate
             Enzyme      Enzyme          Now fits




  Positive           Active Site
 Regulator            Changed
 Positive allosterism - activates the enzyme.
 Negative allosterism - deactivates the enzyme.
Ch106; Chpt 21 Enzymes                              20 - 49
     Inactive            Negative
     Enzyme                            Feedback Control
                         Regulator

        E1                                 End Product
                                            Stops E1

                                  B        C        D

                              A
     E1                  E1           E2       E3
    Active
  Allosteric
   Enzyme



Ch106; Chpt 21 Enzymes                                   20 - 50
                                Cofactors
       Apoenzyme
     •protein portion
         •Inactive
                                  Co2+




        Cofactor         Co2+
   Non protein Group
    need to „activate‟
       apoenzyme
Ch106; Chpt 21 Enzymes                   20 - 54
                                           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



Ch106; Chpt 21 Enzymes                                          20 - 56
                                              Coenzymes
         Organic molecule that temporarily binds to
         apoenzyme in order for it to work




                         +


                             coenzyme
          apoenzyme                        holoenzyme

            Protein          Non-Protein     Total
Ch106; Chpt 21 Enzymes                                  20 - 57
                                         Vitamins are often
                                    converted 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

Ch106; Chpt 21 Enzymes                                            20 - 59
                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.


Ch106; Chpt 21 Enzymes                             20 - 60
Ch106; Chpt 21 Enzymes   20 - 61
                                     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.
Ch106; Chpt 21 Enzymes                            20 - 62
                         Specific enzyme examples

   Let‟s 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.

Ch106; Chpt 21 Enzymes                               20 - 63
                                      Chymotrypsin

    This enzyme is a proteolytic
    enzyme. It cleaves peptide
    bonds.

            H O         H
            | ||        |
    -C-HN - C - C - N - C -
            |       | |       This enzyme only works
            R       H R1      on amino acids containing
                              an aromatic ring.
              Peptide bond    phenylalanine, tyrosine
                              and tryptophan.
Ch106; Chpt 21 Enzymes                                  20 - 64
                         Acetylcholinesterase and
                               nerve 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.
Ch106; Chpt 21 Enzymes                                  20 - 65
                         Acetylcholinesterase and
                               nerve transmission
                                 Presence of
                                 acetylcholine at receptor
                                 causes a flow of sodium
              synaptic           and potassium ions.
              cleft              This causes a muscle
                                 contraction.


                                   acetylcholine
                                   receptor protein

                                 acetylcholinesterase
                                 - destroys excess
                                   acetylcholine
Ch106; Chpt 21 Enzymes                                  20 - 66
                                   Acetylcholinesterase
                            Stick model of
                         acetylcholinesterase.




Ch106; Chpt 21 Enzymes                               20 - 67
                         Acetylcholinesterase and
                               nerve 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.
Ch106; Chpt 21 Enzymes                              20 - 68
                                    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.

Ch106; Chpt 21 Enzymes                                    20 - 69
                               Fibrin




                         Ribbon model
                           of fibrin.




Ch106; Chpt 21 Enzymes              20 - 71
                                 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.

Ch106; Chpt 21 Enzymes                                  20 - 72
              thrombin   antithrombin
Ch106; Chpt 21 Enzymes                  20 - 73
                                        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.
Ch106; Chpt 21 Enzymes                                          20 - 74
                             Defective enzymes
                                    and 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

Ch106; Chpt 21 Enzymes                               20 - 75
                         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).
Ch106; Chpt 21 Enzymes                              20 - 76
                                Phenylketonuria (PKU)

    PKU is one of a family of enzymatic/genetic
    disorders related to phenylalanine metabolism.
       phenylalanine                       tyrosine
                          PKU
          -CH2-CH-COOH              HO-    -CH2-CH-COOH
               |                                |
               NH2        blocked               NH2    albinism



                         alkaptonuria                    melanin
     CH-COO-      O
                  ||                      -CH2-COOH
     ||      +
              CH3C-SCoAblocked
    HC-COO-    acetyl CoA                 Homogentisic acid
    fumarate
Ch106; Chpt 21 Enzymes                                         20 - 77
                         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.
Ch106; Chpt 21 Enzymes                             20 - 78

				
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