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    Enzymes & Biological Catalysis

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    joer13
    Enzymes greatly accelerate biochemical reactions by lowering the activation energy needed to reach the transition state. They do this by tightly binding substrates in a manner that favors the transition state geometry. This promotes the formation of enzyme-substrate complexes and allows enzymes to selectively catalyze specific reactions among many possibilities. The active site provides an optimized environment for reactions through interactions that position substrates, stabilize charged intermediates, and use cofactors and catalytic residues to enhance the electrophilic or nucleophilic nature of reactive groups. Enzymes are also subject to strict regulation and can be inhibited through competitive, uncompetitive or noncompetitive mechanisms.

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    Enzymes & Biological Catalysis

    Uploaded by

    joer13
    39 views64 pages
    Enzymes greatly accelerate biochemical reactions by lowering the activation energy needed to reach the transition state. They do this by tightly binding substrates in a manner that favors the transition state geometry. This promotes the formation of enzyme-substrate complexes and allows enzymes to selectively catalyze specific reactions among many possibilities. The active site provides an optimized environment for reactions through interactions that position substrates, stabilize charged intermediates, and use cofactors and catalytic residues to enhance the electrophilic or nucleophilic nature of reactive groups. Enzymes are also subject to strict regulation and can be inhibited through competitive, uncompetitive or noncompetitive mechanisms.

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    Enzymes Outline

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    Enzymes greatly accelerate biochemical reactions by lowering the activation energy needed to reach the transition state. They do this by tightly binding substrates in a manner that favors the transition state geometry. This promotes the formation of enzyme-substrate complexes and allows enzymes to selectively catalyze specific reactions among many possibilities. The active site provides an optimized environment for reactions through interactions that position substrates, stabilize charged intermediates, and use cofactors and catalytic residues to enhance the electrophilic or nucleophilic nature of reactive groups. Enzymes are also subject to strict regulation and can be inhibited through competitive, uncompetitive or noncompetitive mechanisms.

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    39 views64 pages

    Enzymes & Biological Catalysis

    Uploaded by

    joer13
    Enzymes greatly accelerate biochemical reactions by lowering the activation energy needed to reach the transition state. They do this by tightly binding substrates in a manner that favors the transition state geometry. This promotes the formation of enzyme-substrate complexes and allows enzymes to selectively catalyze specific reactions among many possibilities. The active site provides an optimized environment for reactions through interactions that position substrates, stabilize charged intermediates, and use cofactors and catalytic residues to enhance the electrophilic or nucleophilic nature of reactive groups. Enzymes are also subject to strict regulation and can be inhibited through competitive, uncompetitive or noncompetitive mechanisms.

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    Enzymes & Biological Catalysis

    Enzymes Increase Reaction Rates

    uncatalyzed
    Moving Object ant crawling person walking race horse gallop Bugatti Veyron FA-22 Raptor Space shuttle Photon from sun Speed (mph) 0.06 3 40 253 1500 17,500 6.7 x 108

    catalyzed
    Rate Increase 1 101.6 102.9 103.7 104.4 105.5 108.8

    Enzymes enhance reaction rates 5-17 orders of magnitude

    Enzymes Select Specific Pathways


    Enzymes catalyze one out of many possible reactions
    O

    acetaldehyde

    B
    O

    CO2 ADP Pi HCO3ATP NADH pyruvate NAD+

    OH

    A E

    -OOC

    oxaloacetate

    COO-

    COO-

    NAD+ NADH
    O

    CoASH CO2

    lactate

    COO-

    acetyl-CoA
    SCoA

    4 possible reactions for pyruvate

    Enzymes Allow Reaction Regulation


    O
    -OOC

    ADP Pi
    COO-

    HCO3ATP

    oxaloacetate

    Enzyme-catalyzed reactions are subject to strict regulation

    pyruvate pyruvate carboxylase CoASH NAD+ pyruvate dehydrogenase NADH CO2


    O

    COO-

    SCoA

    acetyl-CoA

    OH
    -OOC

    COOCOO-

    citrate

    Enzyme Classes

    Cofactors

    Mg2+ Zn2+ Cu2+ Fe3+ Ca2+ bind enzyme transiently stably bound to enzyme

    Vitamin-derived Coenzymes

    Enzyme Active Sites


    Active site: special environment that accelerates chemical transformations

    Active Sites Bind Substrates

    Reaction Equilibria and Free Energy

    [B] K eq = [A]
    A B

    G = RT lnK eq
    A quantitative measure of the tendency of a reaction
    28

    Mix A, B

    Measure A, B

    G > 0, reaction unfavored (endergonic) G < 0, reaction favored (exergonic) G = 0, reaction at equilibrium

    G 0
    -28 10-5 1 105

    Keq

    Effect of Enzymes

    Enzymes Lower Energy of Transition State

    Enzymes Lower Energy of Activation

    Enzyme binding to substrate and product critical for rate enhancement

    The ES Complex Weak bonds bind substrate to enzyme.


    Provides much of energy needed to lower G. ES formation accompanied by lower G, termed binding energy (GB).
    Major source of energy used to lower G.

    GB contributes to specificity and catalysis. Weak interactions between E and S are optimized at the transition state.
    Active sites shape is complementary to the geometry of the reaction transition state.

    Formation of ES Complexes

    Lock-and-key model

    Induced-fit model

    Summary of Key Points


    Geometry and chemical make-up of active site responsible for rate enhancement and specificity

    Enzymes greatly accelerate reactions in a highly specific manner

    Acceleration due to reduction in energy needed to reach transition state NO effect on Keq

    Favorable weak interactions between E and S promote ES, lower transition state energy

    Maximal interactions between E and S occur when S assumes the shape of the transition state

    Enzyme Kinetics

    [Vmax ][S] v0 = K m + [S]


    Michaelis-Menton equation

    (when Km >> [S]) (when [S] >> Km)

    (when Km = [S])

    Double-reciprocal Plot Estimates Vmax, Km

    Turnover Number: kcat


    Estimates the rate an enzyme can operate on a per active site basis under conditions when the enzyme is saturated

    kcat = maximum number of molecules of substrate that can be converted to product per second per active site

    V max k cat = ; [ET]

    kcat[ET][S] Vo = Km + [S]

    kcat units: s-1

    Specificity Constant: kcat/Km Under physiologic conditions, most enzymes operate at [S]:Km = 0.01-1.0.
    If Km >> [S], then Specificity constant measures speed of enzyme when [S] low.
    Index of efficiency of intermolecular collisions.
    Diffusion controlled reactions have k~108 .

    Vmax[S] kcat v = [E][S] Km Km


    Units: M-1s-1

    Allows v comparisons of different substrates for a given enzyme.

    Enzyme Inhibition
    Various agents block enzyme activity

    Ibuprofen
    O

    OH

    Types of Inhibition
    Irreversible inhibitors Reversible inhibitors Competitive Uncompetitive Noncompetitive
    Form irreversible complex with E, mechanism-based inactivators

    (aka, noncompetitive)

    Competitive Inhibition

    v0 =

    Vmax [S] K M + [S]

    = 1+

    [I] KI

    is index of inhibitors affinity for E

    Uncompetitive Inhibition
    Vmax [S] v0 = K M + a [S] [I] = 1+ KI

    is index of inhibitors afnity for ES complex

    Noncompetitive (Mixed) Inhibition


    Vmax [S] v0 = K M + [S]

    Table 12-2

    Effect of Ibuprofen on PGG2 [S] 0.5 1.0 1.5 2.5 3.5 Vo 23.5 32.2 36.9 41.8 44.0 +IB 16.67 25.25 30.49 37.04 38.91 1/[S] 2 1 0.67 0.40 0.286 1/Vo 0.043 0.031 0.027 0.024 0.023 +IB 0.06 0.04 0.033 0.027 0.026

    Catalytic Strategies
    Promote favorable geometric configuration and proximity of substrates Guiding principles Enhance partial charges on reactive species Stabilize transition states Stabilize charged reaction intermediates

    Proximity and Orientation Effects


    Enzymes bring reacting species close together. Energy of interaction helps overcome entropy decrease. Reactants bound with orientation that promotes transition state.
    GAPDH in complex with NAD+ and GAP (PDB 1NQO)
    O P -O OOH O H O

    glyceraldehyde-3-phosphate NAD+, Pi

    NADH
    O P -O OOH O O OO O P O-

    1,3-diphosphoglycerate

    Intramolecular v. Intermolecular Reaction Rates

    Intramolecular reactions faster

    General Acid, General Base Catalysis


    Bond rearrangement or breakage requires electron migration Reactive chemical groups: electrophiles, nucleophiles

    O R' O

    O R' O O C R HO R' H

    O C R

    C + R

    H H

    OH

    General Acid Catalyzed Reactions

    General Base Catalyzed Reactions

    Electrostatic Effects
    A xed charge or polar group can stabilize the transition state.
    Positive charged moiety can stabilize transient negative charges.
    O C R1 O R2 R1 O H O H R3 O C O R3

    NH3 OC O R2

    Active sites often hydrophobic.


    Low dielectric constant enhances charge on ionic groups, increases electrostatic interactions.

    tetrahedral transition state

    Enzymatic Nucleophiles, Electrophiles

    A group on an enzyme acts as a nucleophile or electrophile

    Structural Flexibility
    Substrate binding often induces conformational change in enzyme. Overall effects:

    Examples
    Hexokinase
    Glc + ATPGlc-6P + ADP

    Chymotrypsin

    HK

    HK + Glc

    Serine Proteases
    Large family of hydrolases with diverse functions

    O O N+ H2O

    acetylcholine

    acetylcholinesterase
    O OHO N+

    Catalytic triad: Asp, His, Ser


    His

    acetate

    choline
    Asp O OH N N H O Ser

    Active Site is 3-Dimensional Construct

    Asp, His, Ser are not contiguous


    Triad only comes together when protein folds into native structure

    Trypsin

    Specificity Pocket

    Protease chymotrypsin trypsin Elastase

    Specificity Phe/Trp/Tyr-X Lys/Arg-X small neutral (Ala)-X

    General Serine Protease Mechanism

    Figure 11-29 part 5

    Zymogens
    Proteolytic enzymes usually synthesized as inactive precursors Zymogenic form prevents active proteases from digesting tissues that synthesize them

    pancreatic acinar cells

    Trypsinogen: Zymogen of Trypsin


    trypsinogen-specific peptide signal sequence active trypsin

    MKTFIFLALL GANTVPYQVS AAHCYKSGIQ SKSIVHPSYN SRVASISLPT GTSYPDVLKC SNMFCAGYEG GIVSWGSGCA TIASN

    GAAVAFPVDD LNSGYHFCGG VRLGEDNINV SNTLNNDIML SCASAGTQCL LKAPILSDSS GKDSCQGDSG QKNKPGVYTK

    DDKIVGGYTC SLINSQWVVS VEGNEQFISA IKLKSAASLN ISGWGNTKSS CKSAYPGQIT GPVVCSGKLQ VCNYVSWIKQ

    Enteropeptidase or trypsin clips off VDDDDK peptide, activating trypsin

    Trypsin Activation by Ile 16

    N-term Ile NH2 bonds to Asp 194


    Frees up active site to bind substrate

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