2 enzymes & enzyme kinetics.ppt

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The enzyme active site (features) • The catalytic site is relatively small compared with the rest of the enzyme. Why are many enzymes so big then? • The catalytic site is a three- dimensional entity • Substrates are bound to enzymes by multiple weak, non-covalent interactions (electrostatic bonds, hydrogen bonds, van der Waals forces, hydrophobic interactions)

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  • The enzyme active site (features)The catalytic site is relatively small compared with the rest of the enzyme. Why are many enzymes so big then?The catalytic site is a three-dimensional entitySubstrates are bound to enzymes by multiple weak, non-covalent interactions (electrostatic bonds, hydrogen bonds, van der Waals forces, hydrophobic interactions)

  • Ribonuclease

  • Catalytic sites form clefts or crevicesSubstrate molecules bound within cleftWater (unless involved in catalysis) is normally excludedOverall nonpolar character of cleft can enhance binding of substrateCleft may also contain polar residues which may take on catalytic properties within this nonpolar microenvironment (exception to the rule regarding hydrophobic core present in many globular proteins)

  • Active site of cytochrome P-450

  • Active site involves amino acids far apart in the primary sequence of a protein (example: lysozyme)

  • The specificity of binding depends on the precisely defined arrangement of atoms in an active siteEmil Fischer (over 100 years ago): came up with the lock and key hypothesis to describe enzyme-substrate interactions

  • Induced fit model: a more refined model that takes into account the enzyme assumes a complimentary shape to that of its substrate only after substrate binds to the enzyme.More dynamic scenario compared to the lock and key hypothesis

  • Michaelis-Menten model of enzyme kinetics (Vmax & Km)Key element in their model is the existence of the ES complexRate of catalysis (V) increases with increasing [S], where V is defined as the number of moles of product formed per second

  • When enzyme concentrations are constant, V is linearly proportional to [S] WHEN [S] IS SMALL. At high [S] (when S is in vast excess of the [enzyme]), V is nearly independent of [S]

  • The Michaelis-Menten equation

  • Km & VmaxKm = the Michaelis constantDefined as the [substrate] at which the reaction rate is half of its maximal valueUsed to define relative affinity of an enzyme for its substrateThe higher the Km value, the lower the affinity and vice versa

  • Vmax: describes the maximal rate of product formation when [S] is high (i.e., in vast excess of enzyme). Under such conditions all of the existing pool of enzyme active sites are fullFrom Vmax an enzymes turnover number can be determined (expressed as the number of substrate molecules converted into product per unit time)

  • Double-reciprocal (lineweaver-Burk) plotUsed to calculate Km & VmaxAlso used to characterize mechanisms of enzyme inhibition by specific compoundsData expressed as 1/V versus 1/[S]: gives a straight line

  • Calculating Km and Vmax

  • Allosteric enzymes do not conform to Michaelis-Menten kineticsYield a sigmoidal curve on a V versus S plot (not hyperbolic as seen under Michaelis-Menten conditions)Sigmoidal curve indicates cooperative binding (binding of one molecule of S affects affinity and binding of additional S molecules)Regulatory molecules can alter activity of allosteric enzymes

  • Enzyme inhibitionFor enzymes that obey Michaelis-Menten laws, compounds that reversibly inhibit enzyme activity can be kinetically classifiedConsider two general types:Competitive inhibitorsNoncompetitive inhibitors

  • Competitive vs. noncompetitive inhibitors

  • Competitive inhibitorsY intercept the same regardless of whether inhibitor is present or absent, BUT the slope differs between the two lines

  • Competitive inhibitorsDo not alter VmaxIncrease KmCompetitive inhibition can be overcome by increasing substrate concentrationBlock substrate binding to the active site of an enzyme

  • Examples of competitive inhibitorsAlcohol (alcohol dehydrogenase)UpCA (RNase)DHFR inhibitors (DNA metabolic inhibitor of tumors)Sulfa drugs (anti-bacterial drugs)Physiological examples: feedback inhibition, pancreatic trypsin inhibitor

  • Enzyme inhibition & automobile antifreezeEthylene glycol (EG) is a constituent of antifreezeEG not toxic but is converted to oxalic acid which form crystals in the kidneys leading to extensive tissue damage and renal failure

  • First step of conversion of EG to oxalic acid is its oxidation to an aldehyde by alcohol dehydrogenaseThis reaction inhibited by ethanol which competes with EG for binding to the alcohol dehydrogenase

  • Inhibition of RNase by UpCAAn example of atypical competitiveinhibitor:

    UpCA has a verysimilar structureto the genuine substrate, but ischemically unable to undergo reaction.

  • Folate (folic acid)Transformation of folate to tetrahydrofolate catalyzed by dihydrofolate reductase:Competitive inhibitors of dihydrofolate reductase used in cancer treatment (resemble folate, bind ~1000x tighter): eventually leads to synthesis of thymine nucleotides (DNA metabolism)Use of Enzyme inhibitors as anti-cancer drugs:

  • Sulfa DrugsResemble PABA in structureBlocks metabolic activity of bacteria

  • Examples of the Physiological (regulatory) Role of Enzyme Inhibitors

    Feedback inhibition: The end-product of a biochemical pathway is similar to thestarting product and may (competitively) bind to and inhibit one of the enzymesin the pathway:

  • Another example of regulatory competitive inhibition: Inhibitionby Pancreatic Trypsin Inhibitor

  • Noncompetitive inhibitorsPlots converge on the X axis in the presence or absence of inhibitor

  • Noncompetitive inhibitorsDo not alter KmDecrease VmaxNoncompetitive inhibition cannot be overcome by adding excess substrateBind to a site outside of catalytic site of enzyme and act by decreasing the turnover number of an enzyme

  • In noncompetitive inhibition why is Vmax decreased while Km remains unchanged?

  • The inhibitor lowers the concentration of functional enzymeThe remaining uninhibited enzyme behaves like a more dilute solution of that enzyme (assumes [inhibitor] is limiting)In other words, the substrate can still bind to enzyme alone or enzyme complexed with the inhibitor. But only free enzyme will catalyze the reaction.Since the pool of free enzyme is lower in presence of inhibitor, Vmax will also be lower

  • Irreversible Enzyme InhibitorsInhibitor becomes covalently linked to the enzymeAttachment often occurs at the active siteExamples: 5-fluorouracil, DIPF (nerve gas), penicillin

  • Suicide InhibitorsIrreversible enzyme inhibitors Participate in the enzymatic reaction like the substrateAt some point in the reaction they get stuck and become permanently linked to the enzyme.Example: 5-Fluorouracil, a suicide inhibitor which targets thymidylate synthase and is used in cancer treatement.

  • 5-FluorouracilTS cannot catalyze reaction

  • A deadly application of irreversible enzyme inhibitionDIPF (Nerve Gas)DIPF becomes permanently linked to the active-site serine of serine proteasesThe toxic effect comes from inactivation of acetylcholinesteraseThe normal function of this serine protease is to digest the neuromuscular transmitter acetylcholineWhen acetylcholinesterase is inactivated acetylcholine persists. This leads to muscle paralysis and death.

  • Enzyme inhibitors as anti-bacterial drugsPenicillin

  • Most Drugs and toxins are enzyme inhibitors: