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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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