BIOLCHEM 415 1st Edition Lecture 7 Outline of Last Lecture I. Introduction to Enzymes and CofactorsII. Review Thermodynamics with respect to enzymesIII. Active sites and their structuresIV. Different types of enzyme catalysisOutline of Current Lecture V. Enzyme kineticsVI. Reversible and Irreversible inhibition of enzymesVII. Allosteric regulation – binding of a molecule at a site other than the active siteCurrent LectureReactions approach the maximum velocity at high concentrations of substrate- graph has a hyperbolic curveMichaelis-Menten Kinetics- steady-state kinetics- E+S <-> ES -> E+P- once the product is formed the reaction is irreversible- assumes that the concentration of substrate is much greater than the concentration of the enzyme- stability of ES (enzyme substrate complex) defined byKm ≈ (k1+k2)/k1- Vo = Vmax([S]/([S]+Km))- Km = concentration of substrate when reaction is ½ max rateLineweaver Burk Plot1/Vo = 1/Vm + Km/Vm * 1/[S]These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- linearization of the Michaelis-Menten equationKm varies between enzymeskcat : Turnover number- # of reactions catalyzed in the active site per unit time when the enzyme is saturated withkcat/Km- catalytic efficiencyImportant Steady-State Kinetics Points- Km : binding affinity of the enzyme for the substrate- kcat : tells how fast the catalyzed reaction occurs when saturated with substrate- kcat/Km : allows comparison of activities of different enzymesEnzymes are modulated by pH and temperatureInhibitors- interferes with catalyst- reversible or irreversibleReversible Enzyme Inhibitors- competitive inhibitor- competes with substrate for binding in enzyme’s active site- structurally similar to substrate- can increase apparent Km but not Vmax- uncompetitive inhibitor- binds to ES complex- decrease apparent Vmax and the Km- noncompetitive inhibitor- can combine to enzyme or ES complex- doesn’t prevent substrate from binding- slows rate of reaction- decreases the apparent Vmax but not KmIrreversible Inhibitors- modify enzyme or cofactor by forming new covalent bonds- Group Specific Reagents- react with side chains of specific amino acids- ex: Diisopropylphosphofluoridate- forms covalent bonds with hydroxyl groups of reactive serines- Affinity labels- substrates chemically modified to general specific covalent labeling reagents- ex: Tosyl-L-phenylalanine chloromethyl ketone- reactive substrate analog that forms a covalent bond with His57 in catalytic triad of chymotrypsin- Suicide Inhibitor- chemically modified substrate- intermediate covalently modifies active site amino acid- ex: penicillin- β–lactam antibiotic that inhibits the biosynthesis of the cell wall in bacteriaAllosteric Regulation of Enzyme Activity- regulation of enzyme or protein activity by binding of molecule at site other than the active site- exhibit relaxed (R) and Tense (T) state- Aspartate Transcarbamoylase (ATCase)- multi-subunit enzyme - catalyzes first committed step in biosynthesis of pyrimidine nucleotides- T state: weak binding affinity for substrate aspartate- R state: strong binding affinity for aspartate- in absence of a substrate, enzyme exists in the T
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