chapter 11 enzymes final

Download Chapter 11 Enzymes FINAL

Post on 21-Dec-2015




0 download

Embed Size (px)


i need a document



    Fourth EditionDonald Voet - Judith G. Voet - Charlotte W. Pratt

    Chapter 11:Enzyme Catalysis


  • Enzyme Catalysis

    Enzyme activity and classification

    Enzyme specificity and cofactors

    Transition-State Diagrams

    Catalytic Mechanisms


    Serine Proteases


  • Enzymes Enzymes differ from ordinary chemical catalyst in several important respects:

    Higher reactions rates: The rates of enzymatically catalyzed reactions are typically several of orders of magnitude greater than those of the corresponding uncatalyzed reactions.

    Milder reactions conditions: Enzymatically catalyzed reactions occur under relative mild conditions (below 100C, atmospheric pressure, and nearly neutral pH)

    Greater reaction specificity Capacity for regulation: The catalytic activities of many enzymes

    vary in response to the concentrations of substances other than their substrates.


  • Enzymes


  • Reaction Coordinate Diagrams

    reactants (substrate, S) products (P) enzyme (E) transition states G

    - spontaneous rxn activation energy



    E + S ES E + P


  • Reaction Coordinate Diagrams

    Catalysts act by providing a reaction pathway with a transition state whose free energy is lower than in the uncatalyzed reaction.

    lowers the activation energy does not change Grxn


    E + S ES E + P


  • Reaction Coordinate DiagramsChemical reactions commonly consist of several steps. For a two-step reaction such as:

    A I P

    there are two transition sates and two activation energy barriers.

    The step with the highest transition state acts as a bottleneck and is said to be the rate-limiting step.


  • Enzyme ClassificationEnzymes are commonly named by appending the suffix -ase to the name of the enzyme's substrate or to a phrase describing the enzyme's catalytic action.

    urease catalyzes the hydrolysis of urea

    alcohol dehydrogenase catalyzes the oxidation of alcohols by removing hydrogen.

    There are six major classes of enzymatic reactions.


    Nile Dresser

    Nile Dresserknow these

  • Enzyme SpecificitySubstrate-binding site consists of an indentation or cleft on the surface of an enzyme molecule that is complementary in shape to the substrate (geometric complementarity).

    Amino acids residues that form the binding site are arranged to specifically attract the substrate (electronic complementarity).

    Enzymes act on specific substrates via: van der Waals electrostatic hydrogen bonding hydrophobic interaction

    Nearly all enzymes that participate in chiral reactions are absolutely stereospecific.


  • Enzyme SpecificityEnzymes vary in the degree of geometric specificity. A few enzymes are absolutely specific for only one compound.

    Most enzymes, however, catalyze the reactions of a small range of related compounds, but with different efficiencies.

    Alcohol dehydrogenase catalyzes the oxidation of ethanol to acetaldehyde faster than it oxidizes methanol to formaldehyde (or isopropanol to acetone).

    Digestive enzymes


  • Cofactors

    Cofactors may be: metal ions (Cu2+, Fe3+, Zn2+) organic molecules (coenzymes)

    - cosubstrates: NAD+, NADP+

    - prosthetic groups are permanently associated with their protein (heme group in myoglobin, hemoglobin and cytochromes)

    A catalytically active enzyme-cofactor complex is called a holoenzyme.

    An inactive protein resulting from the removal of a holoenzymes cofactor is called an apoenzyme.


  • How do Enzymes Work?Catalysis can occur through proximity and orientation effects. By simply binding their substrates, enzymes facilitate their catalyzed reactions in 4 ways:

    Enzymes bring substrates into contact with the catalytic groups. Enzymes bind their substrates in the proper orientation. Charged groups may help stabilize the transition state of the

    reaction (electrostatic catalysis). Enzymes freeze out the relative translational and rotational motions

    of their substrates and catalytic groups.


  • How do Enzymes Work?

    An enzyme may bind the transition state of the reaction it catalyzes with greater affinity that its substrate or products.

    enzymes that preferentially bind the transition state structure increase its concentration and therefore increase the reaction rate.

    - rationale for drug design and enzyme inhibitors


  • Catalytic MechanismsThe types of catalytic mechanisms that enzymes employ have been classified as:

    Acid-base catalysis Metal ion catalysis Covalent catalysis

    Proximity and orientation effects Preferential binding of the transition state complex


  • Acid-Base CatalysisGeneral acid catalysis is a process in which proton transfer from an acid lowers the free energy of a reactions transition state.

    hydrolysis of peptide bonds phosphate group reactions

    The side chains of Asp, Glu, His, Cys, Tyr, and Lys have pKs in or near physiological pH range, which permits them to act as acid and/or base catalysts.

    activity is sensitive to pH


  • Effects of pH on ActivityMost enzymes are active within only a narrow pH range (5-9).

    binding of substrate to enzyme the ionization states of the amino acid residues the ionization of the substrate the variation of protein structure

    Inflection point on the curve often provides valuable clues to the identities of the amino acid residues essential for enzymatic activity.

    pK of ~ 4: Asp or Glu pK of ~6: His pK of ~10: Lys


  • RNase ARNase A is an example of enzymatically mediated acid-base catalysis.

    secreted by the pancreas into the small intestine

    hydrolyzes RNA to its component nucleotides

    120 amino acids pI = 9.6


  • His 12 (acting as a base) abstracts a proton from an RNA 2-OH group.This promotes its nucleophilic attack on the adjacent phosphorous atom.His 119 acts as an acid.


  • After leaving group departs, water enters the active site, and the 2,3-cyclic intermediate is hydrolyzed.

    H12 now acts as an acid and H119 as a base.19

  • Metal Ion CatalysisNearly one-third of all known enzymes require metal ions for catalytic activity.

    metalloenzymes contain tightly bound metal ions (Fe2+, Fe3+, Cu2+, Mn2+, Co2+)

    Na+, K+, Ca2+ play a structural role rather than a catalytic role Mg2+ and Zn2+ may be either structural or catalytic

    Metal ion cofactors: bind to substrates to orient them properly mediate oxidation-reduction reactions electrostatically stabilize or shield negative charges


  • Metal Ion CatalysisCarbonic anhydrase:

    catalyzes the rapid conversion of:

    CO2 + H2O HCO3- + H+ contains an essential Zn2+ ion (prosthetic

    group) tetrahedrally coordinated by three His side chains, and 1 H2O molecule

    Zn2+ ion polarizes a water molecule to form OH-, which nucleophilically attacks the substrate CO2 to yield HCO3-.


  • Covalent CatalysisCovalent catalysis accelerates reaction rates through the transient formation of a catalyst-substrate covalent bond.

    serine proteases: acyl-serine intermediate protein kinases and phosphatases: phospho-amino acid interm.

    nucleophilic catalysis: covalent bond is formed by the reaction of a nucleophile group on the catalyst with an electrophilic group on the substrate.

    - side chains of His, Cys, Asp, Lys and Ser can participate in covalent catalysis by acting as nucleophiles.


  • Covalent CatalysisThe nucleophilicity of a substance is closely related to its basicity.

    biological nucleophiles are negatively charged or contain unshared electron pairs

    biological electrophiles include positively charged groups, contain unfilled valence electron shell, or contain an electronegative atom.


  • LysozymeLysozyme is an enzyme that destroys bacteria cell walls.

    hydrolyzing the (14) glycosidic linkage from NAM to NAG in cell wall peptidoglycans.

    occurs widely in the cells of vertebrates- probably functions as bactericidial agent

    example of covalent catalysis and acid-base catalysis.


  • Bacterial Cell Wall


  • LysozymeLysozyme is an enzyme that destroys bacteria cell walls.

    HEW lysozyme in complex with NAG

    Glu 35 and Asp 52 catalyze the hydrolysis of the glycosidic linkage

    - Asp negative charge side chain functions to electronically stabilize the intermediate

    - Glu acts as an acid catalyst


  • 27

  • Serine ProteasesSerine proteases are a group of proteolytic enzymes, which include digestive enzymes and specialized proteins that participate in development, blood clotting, inflammation, etc.

    Serine in their active site

    Digestive Enzymes: synthesized by the pancreas and secreted into the small intestine. catalyze the hydrolysis of peptide bonds (with different specificities) chymotrypsin: specific for a bulky hydrophobic residue (F, Y, W) trypsin: specific for a positively charged residue (R, K) elastase: specific for a small neutral residue (A, G, V)


    Nile Dresser

  • Identifying Important Amino AcidsChemical labeling studies helped identify chymotrypsins catalytically important groups.

    Diisopropylphosphofluoridate (DIPF): diagnostic test for the presence of the

    active site Ser of serine proteases irreversibly inactivates the enzyme reacts only with Ser 195 of

    chymotrypsin, thereby demonstrating