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Properties of Enzymes

Properties of Enzymes. Catalyst - speeds up attainment of reaction equilibrium Enzymatic reactions - 10 3 to 10 17 faster than the corresponding uncatalyzed reactions Substrates - highly specific reactants for enzymes. Properties of Enzymes.

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Properties of Enzymes

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  1. Properties of Enzymes • Catalyst - speeds up attainment of reaction equilibrium • Enzymatic reactions - 103 to 1017 faster than the corresponding uncatalyzed reactions • Substrates- highly specific reactants for enzymes

  2. Properties of Enzymes Stereospecificity- many enzymes act upon only one stereoisomer of a substrate Reaction specificity - enzyme product yields are essentially 100% (there is no formation of wasteful byproducts) Active site - where enzyme reactions take place

  3. Types of Enzymes • Oxidoreductases (dehydrogenases) • Transferases • Hydrolases • Lyases • Isomerases • Ligases (synthetases)

  4. 1.Oxidoreductases (dehydrogenases)Catalyze oxidation-reduction reactions

  5. 2.Transferases • Catalyze group transfer reactions

  6. 3. Hydrolases • Catalyze hydrolysis reactions where water is the acceptor of the transferred group

  7. 4. Lyases Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination (Synthases catalyze the addition to a double bond, the reverse reaction of a lyase)

  8. 5. Isomerases • Catalyze isomerization reactions

  9. 6.Ligases (synthetases) • Catalyze ligation, or joining of two substrates • Require chemical energy (e.g. ATP)

  10. Inhibitor(I) binds to an enzyme and prevents formation of ES complex or breakdown to E + P • Inhibition constant(Ki) is a dissociationconstant EI E + I • There are three basic types of inhibition: Competitive, Uncompetitive and Noncompetitive • These can be distinguished experimentally by their effects on the enzyme kinetic patterns Enzyme Inhibition (Reversible)

  11. Reversible enzyme inhibitors (a) Competitive. S and I bind to same site on E (b) Nonclassical competitive. Binding of S at active site prevents binding of I at separate site. Binding of I at separate site prevents S binding at active site.

  12. Reversible enzyme inhibitors (c) Uncompetitive.I binds only to ES (inactivates E) (d) Noncompetitive.I binds to either E or ES to inactivate the enzyme

  13. Competitive Inhibition • Inhibitor binds only to freeenzyme (E) not (ES) • Substrate cannot bind when I is bound at active site(S and I “compete” for the enzyme active site) • Competitive inhibitors usually resemblethesubstrate

  14. Benzamidine competes with arginine for binding to trypsin

  15. Irreversible Enzyme Inhibition • Irreversible inhibitors form stablecovalentbonds with the enzyme (e.g. alkylation or acylation of an active site side chain) • There are many naturally-occurring and synthetic irreversible inhibitors • These inhibitors can be used to identify the amino acid residues at enzyme active sites • Incubation of I with enzyme results in loss of activity

  16. Covalent complex with lysine residues • Reduction of a Schiff base forms a stable substituted enzyme

  17. Inhibition of serine protease with DFP • Diisopropyl fluorophosphate (DFP) is an organic phosphate that inactivates serine proteases • DFP reacts with the active site serine (Ser-195) of chymotrypsin to form DFP-chymotrypsin • Such organophosphorous inhibitors are used as insecticides or for enzyme research • These inhibitors are toxic because they inhibit acetylcholinesterase (a serine protease that hydrolyzes the neurotransmitter acetylcholine)

  18. Affinity labels for studying enzyme active sites • Affinity labelsare active-site directed reagents • They are irreversible inhibitors • Affinity labels resemble substrates, but contain reactive groups to interact covalently with the enzyme

  19. Site-Directed Mutagenesis Modifies Enzymes • Site-directed mutagenesis(SDM) can be used to test the functions of individual amino acid side chains • One amino acid is replaced by another using molecular biology techniques • Bacterial cells can be used to synthesize the modified protein

  20. Practical applications of SDM • SDM is also used to change the properties of enzymes to make them more useful • Subtilisin protease was made more resistant to chemical oxidation by replacing Met-222 with Ala-222 (the modified subtilisin is used in detergents) • A bacterial protease was made more heat stable by replacing 8 of 319 amino acids

  21. Regulation of Enzyme Activity

  22. Two Methods of regulation • (1) Noncovalentallosteric regulation • (2) Covalentmodification • Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site • Allostericinhibitors or activators bind to this site and regulate enzyme activity via conformational changes

  23. General Properties of Allosteric Enzymes 1. Activities of allosteric regulator enzymes are changed by inhibitors and activators (modulators) 2. Allosteric modulators bind noncovalently to the enzymes that they regulate 3. Regulatory enzymes possess quaternarystructure (continued next slide)

  24. General Properties of Allosteric Enzymes 4. There is a rapid transition between theactive (R) and inactive(T) conformations 5. Substrates and activators may bind only to the R state while inhibitors may bind only to the T state

  25. Rapid transition exists between R and T • Addition of S increases concentration of the R state • Addition of I increases concentration of the T state • Activator molecules bind preferentially to R, leading to an increase in the R/T ratio

  26. Two Theories of Allosteric Regulation • Concerted theory - (symmetry-driven theory). • Only 2 conformations exist: R and T ( symmetryisretained in the shift between R and T states) • Subunits are either all R or all T • R has high affinity for S, T has a low affinity for S • Binding of S shifts the equilibrium toward all R state • Binding of I shifts the equilibrium toward all T state

  27. Sequential Theory (ligand-induced theory) • A ligand may induce a change in the structure of the subunit towhichitbinds • Conformational change of one subunit may affect the conformation of neighboring subunits • A mixture of both R (high S affinity) and T (low S affinity) subunits may exist (symmetry does not have to be conserved)

  28. (a) Concertedmodel: subunits either all T state or all R state (b) Sequentialmodel: Mixture of T subunits and R subunits is possible. Binding of S converts only that subunit from T to R

  29. Conformational changes during O2 binding to hemoglobin • Oxygen binding to Hb has aspects of both the sequential and concerted models

  30. Regulation by Covalent Modification • Interconvertible enzymes are controlled by covalentmodification • Converter enzymes catalyze covalentmodification • Converter enzymes are usually controlled themselves by allostericmodulators

  31. Pyruvate dehydrogenaseregulation • Phosphorylation stabilizes the inactivestate (red) • Dephosphorlyation stabilizes the activestate (green)

  32. Kinetic Experiments Reveal Enzyme Properties Chemical Kinetics • Experiments examine the amount of product(P) formed per unit of time (D[P] / Dt) • Velocity(v) - the rate of a reaction (varies with reactant concentration) • Rate constant(k)- indicates the speed or efficiency of a reaction

  33. First order rate equation • Rate for nonenzymatic conversion of substrate (S) to product (P) in a first order reaction: (k is expressed in reciprocal time units (s-1)) D[P] /Dt = v = k[S]

  34. For reactions: S1 + S2 P1 + P2 • Rate is determined by the concentration of both substrates • Rate equation: v = k[S1]1[S2]1 Second order reaction

  35. Pseudo first order reaction • If the concentration of one reactant is so high that it remains essentially constant, reaction becomes zeroorderwithrespecttothatreactant • Overall reaction is then pseudo first-order v = k[S1]1[S2]0 = k’[S1]1

  36. E + SESE + P Enzyme Kinetics • Enzyme-substrate complex(ES) - complex formed when specific substrates fit into the enzyme active site • When [S] >> [E], every enzyme binds a molecule of substrate (enzyme is saturated with substrate) • Under these conditions the rate depends only upon [E], and the reaction is pseudo-first order

  37. Effect of enzyme concentration [E] on velocity (v) • Fixed, saturating [S] • Pseudo-first order enzyme-catalyzed reaction

  38. k1 k2 E + S ES E + P k-1 Initial velocity (vo) • Velocity at the beginning of an enzyme-catalyzed reaction is vo(initial velocity) • k1 and k-1 represent rapid noncovalent association /dissociation of substrate from enzyme active site • k2 = rate constant for formation of product from ES

  39. The Michaelis-Menten Equation • Maximum velocity(Vmax) is reached when an enzyme is saturatedwithsubstrate (high [S]) • At high [S] the reaction rate is independent of [S] (zero order with respect to S) • At low [S] reaction is firstorder with respect to S • The shape of a vo versus [S] curve is a rectangular hyperbola, indicating saturation of the enzyme active site as [S] increases

  40. Plots of initial velocity (vo) versus [S] (a) Each vo vs [S] point is from one kinetic run (b) Michaelis constant (Km) equals the concentration of substrate needed for 1/2 maximum velocity

  41. The Michaelis-Menten equation • Equation describes vo versus [S] plots • Kmis the Michaelis constant Vmax[S] vo = Km + [S]

  42. Derivation of the Michaelis-Menten Equation • Derived from • (1) Steady-state conditions: • Rate of ES formation = Rate of ES decomposition • (2) Michaelis constant: Km = (k-1 + k2) / k1 • (3) Velocity of an enzyme-catalyzed reaction (depends upon rate of conversion of ES to E + P) • vo = k2[ES]

  43. The Meanings of Km • Km = [S] when vo = 1/2 Vmax • Km@ k-1 / k1 = Ks (the enzyme-substrate dissociation constant) when kcat << either k1 or k-1 • The lower the value of Km, the tighter the substrate binding • Km can be a measure of the affinity of E for S

  44. Catalytic constant(kcat) - first order rate constant for conversion of ES complex to E + P • kcat most easily measured when the enzyme is saturated with S • Ratio kcat /Km is a secondorderrateconstant for E + S E + P at low [S] concentrations Kinetic Constants Indicate Enzyme Activity and Specificity

  45. Meanings of kcat and kcat/Km

  46. Measurement of Km and Vmax The double-reciprocal Lineweaver-Burk plot is a linear transformation of the Michaelis-Menten plot (1/vo versus 1/[S])

  47. Competitive inhibition: (a) Kinetic scheme. (b) Lineweaver-Burk plot

  48. Uncompetitive inhibition

  49. Noncompetitive inhibition

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