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  • Unit 3

    Enzymes. Catalysis and enzyme kinetics.

  • 3.1. Characteristics of biological catalysts. Coenzymes, cofactors, vitamins Enzyme nomenclature and classification 3.2. Enzyme catalysis. Transition state Active site Enzyme-substrate complex Factors involved in enzyme catalysis 3.3. Enzyme kinetics. Steady-state assumption and Michaelis-Menten equation Factors affecting the enzymatic activity Enzymatic inhibition • Reversible inhibition • Irreversible inhibition 3.4. Enzyme regulation. Allosteric behaviour Covalent modification Proteolysis

    OUTLINE

  • What characteristics features define enzymes?

    • High catalytic power: ratio of the catalysed rate to the uncatalysed rate of the reaction = 106-1020

    • Enzymes are recover after each catalytic cycle.

    • High specificity: (even stereospecifivity)

    • Regulation

    The biological catalysts are: – Proteins (enzymes) – Catalytic RNA (ribozymes)

    3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

  • • It converts 6x105 molecules per second

    • 107 times faster than the uncatalysed reaction

    Ejemplos de reacciones catalizadas

    • 1011 times faster than the uncatalysed reaction • The specificity depends on the R1 group.

    Protease Carbonic anhydrase

    3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

  • Nonprotein components required for the enzymatic activity: cofactor – Apoenzyme + cofactor = holoenzyme – Two types of cofactors: • Metal ions: Mg2+, Zn2+, Cu2+, Mn2+, ... • Coenzymes: small organic molecules synthesised from vitamins. Prosthetic groups: tightly bound coenzymes Cofactors deficiency promotes some health problems.

    COFACTORS, COENZYMES AND VITAMINS

    3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

  • 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

    COFACTORS, COENZYMES AND VITAMINS

  • 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

    COFACTORS, COENZYMES AND VITAMINS

  • Nº Class Reaction Examples

    1 Oxidoreductases Oxidation-reduction reactions Glucose oxidase (EC 1.1.3.4)

    2 Transferases Transfer of functional groups Hexokinase (EC 2.7.1.2)

    3 Hydrolases Hydrolysis reactions Carboxipeptidase A (EC 3.4.17.1)

    4 Lyases Addition to double bonds Piruvate decarboxylase (EC 4.1.1.1)

    5 Isomerases Isomerisation reactions Malate isomerase (EC 5.2.1.1)

    6 Ligases Formation ob bonds (C-C, C-S, C- O and C-N) with ATP cleavage

    Piruvate carboxylase (EC 6.4.1.1)

    ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

  • Traditional Nomenclature urease: urea hydrolysis amylase: starch hydrolysis DNA polymerase: Nucleotides polymerization • Trivial designations (Ambiguity) Systematic Nomenclature (identify the substrate and the reaction) ATP + D-glucose → ADP + D-glucose 6-phosphate ATP: D-hexose 6-phosphotransferase hexokinase (traditional nomenclature)

    ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

  • Carboxipeptidase A (peptidyl-L-amino acid hydrolase) EC 3.4.17.1 Class: 3 → Hydrolases. Subclass: 4 → peptide bond 17 → metallocarboxypeptidases. Entry number: 1

    A series of four number serves to specify a particular enzyme. The numbers are preceded by the letters EC (enzyme commission). First number: class Second number: subclass (electron donors, type of substrate, etc.) Third number: characteristics of the reaction (functional groups, etc.) Fourth number: order of the individual entries

    ENZYME NOMENCLATURE AND CLASSIFICATION 3.1. CHARACTERISTICS OF BIOLOGICAL CATALYSTS

  • The conversion of S to P occurs because a fraction of the S molecules has the energy necessary to achieve a reactive condition known as the transition state (S-P intermediate)

    Enzymes (catalysts) work by lowering the free energy of activation related to the transition state

    A-B + C

    A….B….C

    A + B-C

    Ej. A-B + C A + B-C

    Transition state

    3.2. ENZYME CATALYSIS

  • Substrate binds at the active site of the enzyme through relatively weak forces (chymotrypsin)

    Specificity Catalytic power

    Active site

    3.2. ENZYME CATALYSIS

  • Lock and key theory (Fisher, 1890)

    Induced fit theory (Koshland y Neet, 1968)

    Enzyme-substrate complex interactions

    3.2. ENZYME CATALYSIS

  • Glucose induced conformational change of hexokinase

    D-glucose

    (a) Unligaded form of hexoquinase and free glucose

    (b) Conformation of hexokinase with glucose bound

    Enzyme-substrate complex interactions

    3.2. ENZYME CATALYSIS

  • FACTORS INVOLVED IN ENZYME CATALYSIS

    • Proximity and orientation

    • Surface phenomena

    • Bounds tension

    • Presence of reactive groups

    3.2. ENZYME CATALYSIS

  • Proximity and orientation

    FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

  • FACTORS INVOLVED IN ENZYME CATALYSIS

    Bounds tension

    3.2. ENZYME CATALYSIS

  • Mechanisms of catalysis

     General acid-base catalysis: proton transference in the transition state (from or towards the substrate)

     Covalent catalysis: transitory covalent bond between enzyme and substrate

    Metal ion catalysis: it acts as electrophilic catalysts, it promotes redox reactions, it stabilised charges, the polarity of certain bounds can change because of the metals…

    Presence of reactive groups FACTORS INVOLVED IN ENZYME CATALYSIS

    3.2. ENZYME CATALYSIS

  • FACTORS INVOLVED IN ENZYME CATALYSIS

    3.2. ENZYME CATALYSIS

  • General acid-base catalysis and covalent catalysis: protease

    Presence of reactive groups

    FACTORS INVOLVED IN ENZYME CATALYSIS

    3.2. ENZYME CATALYSIS

  • Enolase General acid-base catalysis and metal ion catalysis

    FACTORS INVOLVED IN ENZYME CATALYSIS 3.2. ENZYME CATALYSIS

  • It is the analysis of the velocity (or rate) of a chemical reaction

    catalysed by an enzyme, and how the velocities can change on the

    basis of environmental parameters modifications.

    WHAT DO YOU HAVE TO KNOW? • How the rate of an enzyme-catalysed reaction can be defined in a mathematical way • Velocity units • What is the order of a reaction (first-order reaction/second order reaction?

    3.3. ENZYME KINETICS

  • Hypothetical enzyme catalyzing: SP The rate of the reaction decreased when S is converted into P. Initial velocity: slope of tangent to the line at time 0

    The rate of a enzymatic reactions depends on the substrate concentration

    3.3. ENZYME KINETICS

  • 3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the substrate concentration

  • Michaelis-Menten equation describes a curve known as a rectangular hyperbola

    The velocity of the product formation is:

    [ES]kv 2=

    [ES] depends on: the velocity of ES formation from E + S the velocity of its dissociation to regenerate E+S or to form E + P.

    ][][][ 211 ESkESkS[E]kdt d[ES]

    −−= −

    STEADY-STATE ASSUMPTION AND MICHAELIS-MENTEN EQUATION

    E + S ES E + P k1

    k-1

    k2

    3.3. ENZYME KINETICS

  • 0 Time

    Early stage ES formation

    Steady state [ES] is constant

    Steady-state Under experimental conditions [S]>>>[E]. The [ES] quickly reaches a constant value in such dynamic system, and remains constant until complete P formation: Steady State assumption

    3.3. ENZYME KINETICS

  • ][][][ ESEE T +=

    ])[(]][[][][ 2111 ESkkSESkSEk T +=− −

    KM, Michaelis constant

    ])[][(][][ 2111 ESkkSkSEk T ++= −

    211

    1

    ][ ][][][ kkSk

    SEkES T ++

    = −

    121 /)(][ ][][][

    kkkS SEES T ++

    = −

    M

    T

    KS SEES

    + =

    ][ ][][][

    M

    T

    KS SEkv

    + =

    ][ ][][2

    Maximal velocity is obtained when the enzyme is saturated: [E]T=[ES]

    T[E]kV 2max =

    [ES]kv 2=

    MKS SVv

    + =

    ][ ][max

    Michaelis-Menten Equation

    1

    21

    k kkK M

    + = −

    Steady-state

    3.3. ENZYME KINETICS

    ] [ ] [ ] ][ [ , 0 ] [ 2 1 1 ES k ES k S E k dt ES d + = = − so

  • 3.3. ENZYME KINETICS

  • What does KM mean?

    1

    21

    k kkK M

    + = −

    MKS SVv

    + =

    ][ ][max When [S]=KM, v=Vmax/2

    KM is the substrate concentration that gives a velocity equal to one—half the maximal velocity. Units of molarity. It indicates how efficient in an enzyme selecting substrates (specificity) Usually KM is used as a parameter to estimate the affinity of an enzyme for their substrates. KM is similar to the ES dissociation constant when k2

  • 3.3. ENZYME KINETICS The rate of a enzymatic reactions depends on the

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