9,10.Enzymes, Basic Concepts and Kinetics

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    ENZYMES: BASIC CONCEPTS AND KINETICS

    1. Enzyme characteristics1) immense catalytic power

    - 107 - 1017 times faster than the uncatalyzed one

    2) specificity- proteolytic enzymes

    - subtilisin: no specificity

    - trypsin: lysine and arginine

    - thrombin: Arg-Gly in specific peptide sequence

    - DNA polymerase: insertion of wrong nucleotides, 10-6

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    2. Cofactors1) holoenzyme

    - apoenzyme + cofactor

    2) cofactor

    - small molecules required for the catalytic activity of enzymes- metal ions

    - coenzymes: small organic molecules

    - prosthetic group: tightly bound coenzyme

    - loosely bound coenzyme: like substrate

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    3. Classification of enzymes1) oxidation and reduction- oxidoreductase: lactate dehydrogenase

    2) group transfer

    - transferase: nucleotide monophosphate kinase

    3) hydrolysis

    - hydrolase: chymotrypsin4) addition or removal of groups to form double bonds

    - lyase: fumarase

    5) isomerization

    - isomerase: triose phosphate isomerase

    6) ligation of two substrates at the expense of ATP hydrolysis- ligase: aminoacyl-tRNA synthetase

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    4. Enzyme-substrate complex1) active site

    - binding site for the substrates

    2) key features of active sites

    - small part

    - three dimensional entity

    - multiple weak attraction: -3 -12 kcal/mol (10-2 - 10-8)- clefts or crevices: nonpolar microenvironment

    3) specificity of binding

    - lock and key

    - induced fit

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    5. Michaelis-Menten mechanismk1 k2

    E + S ES E + P

    k-11) the 1st step

    - ES complex formation

    - reversible

    2) the 2nd step

    - irreversible3) two assumptions

    - steady state: d[ES]/dt 0

    - initial velocity: [P] = 0

    4) V0 = k2[E]T[S]/([S]+KM): KM = (k-1 + k2)/k1, Vmax = k2[E]T

    5) Lineweaver-Burk plot- 1/V0 = 1/Vmax + KM/Vmax1/[S]

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    6. Significance ofKM and Vmax1) KM: the concentration of substrate at which half the active sites

    are filled

    - fES= [ES]/[E]T = [S]/([S]+KM)2) KM = (k-1 + k2)/k1, k-1k2, KM= k-1/k1- the dissociation constant of ES complex

    - binding strength of substrates for the enzyme

    3) Vmax: velocity when the enzyme is fully saturated with substrate

    - catalytic efficiency

    - Vmax = k2[E]T- k2: the turnover number

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    4) kcat

    /KM

    - [S] KM, V0 = k2/KM [E][S]

    - [E] [E]T

    - V0 = k2/KM [E]T[S] = Vmax/KM[S]- k2/KM = k2k1/(k-1+k2) < k1- -d[S]/dt = k1[E][S]k-1[ES]

    - diffusion-controlled encounter: 108 - 109 M-1S-1- the turnover number for enzymes having more complex pathways: kcat

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    7. Inhibition1) competitive inhibition

    - competitive inhibitors

    - EI complex

    - 1/V0 = 1/Vmax + (1 + [I]/Ki)KM/Vmax1/[S]

    2) noncompetitive inhibition

    - noncompetitive inhibitors

    - EI, ESI complex

    - 1/V0 = (1 + [I]/Ki)1/Vmax + (1 + [I]/Ki)KM/Vmax1/[S]3) uncompetitive inhibition

    - uncompetitive inhibitors

    - ESI complex

    - 1/V0 = (1 + [I]/Ki)1/Vmax + KM/Vmax1/[S]

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    4) irreversible inhibitor

    - group-specific reagents: iodoacetate, diisopropylphosphofluoridate(DIPF)

    - affinity label (reactive substrate analog): tosyl-L-phenylalaninechloromethyl ketone (TPCK), bromoacetol phosphate

    - suicide inhibitor (mechanism based inhibitors): N,N-dimethylpropargylamine monoamine oxidase (target enzyme fordrugs to Parkinson disease

    5) penicillin

    - bacterial cell wall synthesis- glycopeptide transpeptidase

    - acyl-enzyme intermediate

    - penicilloyl-enzyme complex

    - -lactam ring: a transition-state analog of acyl-D-Ala-D-Ala substrate

    - suicide inhibitor: mechanism-based inhibitor6) catalytic antibody (abzyme)

    - antibodies against the transition state analog

    - stabilization of transition states by specific binding interaction

    - low catalytic powers: other catalytic groups besides binding

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    8. Nomenclature for multireactant kinetics

    1) substrates, products- hydrogen ion: normally not a substrate

    - substrate number: uni, bi, ter, quad

    - product number: uni, bi, ter, quad

    2) sequential: binding of all substrates before release of products

    - ordered- random

    - Theorell-Chance: no central complex

    3) ping pong: release of products before binding of all substrates

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    9. Kinetic parameters for multireactant enzymes1) two substrates/two products reaction

    - [B]: fixed

    - [A]: a variable substrate- [B]: a changing fixed variable substrate

    - ordered sequential

    1/V0 = 1/V1(1+Kb/[B]) + Ka/V1(1 + KiaKb/Ka[B])(1/[A])- ping pong

    1/V0

    = 1/V1(1+K

    b/[B]) + K

    a/V

    1(1/[A])

    2) notation for mechanism

    - order sequential

    A B P Q

    E EA AB EQ E

    EPQ

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    - ping pong

    A P B Q

    E EA F FB E

    FP EQ

    3) low concentrations of substrates

    - V0 = V1/Ka[A]- V1/Ka : efficiency of the reaction of E with A- relations between addition points of two substrates

    - reversible: change of slope- irreversible: no change of slope

    - irreversible: product release, substrate saturation

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    10. product inhibition

    1) relations between addition points of a variable substrate and aproduct

    2) reversible: noncompetitive

    3) irreversible: uncompetitive

    4) same enzyme: competitive

    11. non- Michaelis-Menten enzymes1) Cooperative enzymes

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