Enzymes Part 2-3 (1) PDF - Ross University

Cellular Biology & Homeostasis ENZYMES - Part 2 VP Summer 2023 Clara Camargo, DVM LEARNING OBJECTIVES  Define enzyme kinetics and describe its relevance  Explain Michaelis-Menten Kinetics (Vmax, Km, conclusions and curve)  Understand the function of the Lineweaver-Burk plot, explain 1/Vmax and...

Cellular Biology & Homeostasis ENZYMES - Part 2 VP Summer 2023 Clara Camargo, DVM LEARNING OBJECTIVES  Define enzyme kinetics and describe its relevance  Explain Michaelis-Menten Kinetics (Vmax, Km, conclusions and curve)  Understand the function of the Lineweaver-Burk plot, explain 1/Vmax and 1/Km interpretation  Explain the concepts of enzyme inhibition: irreversible vs reversible, competitive and noncompetitive  Explain competitive and non-competitive inhibitors using the Michaelis-Menten enzyme reaction curve and the Lineweaver-Burk plot  List some examples of enzyme inhibitors ENZYME KINETICS The study of the chemical reactions that are catalyzed by enzymes. Studying enzyme kinetics allows for the understanding of how each enzyme works. o The enzyme’s catalytic mechanism o The control of enzyme activity o Its role in metabolism o Possible inhibition by drugs, agonists or antagonists Enzymes Part 2 Enzymes Part 2 MICHAELIS-MENTEN KINECTS Equation describes how reaction velocity varies with substrate concentration  single substrate enzyme kinetics! An enzyme reversibly combines with its substrate to form an ES complex that yields a product P, releasing the free enzyme. Leonor Michaelis (1875-1949) Maud Menten (1879-1960) From: carnotcycle WordPress.com Assumptions/conditions: Relative concentration of enzyme and substrate [S] > > [E] → % of total substrate bound by the enzyme at any one time is small Steady-state: ES complex does not change with time. Rate [ES] formation = [ES] disassociation Initial velocity (V0): reaction is measured as soon as enzyme and substrate are mixed. Concentration of P is very small. Rate of P ES can be ignored Total enzyme concentration [E] does not change over time. Michaelis-Menten kinetics Michaelis Menten kinetics Km Michaelis-Menten constant Is characteristic of an enzyme and its particular substrate and reflects the affinity of the enzyme for that substrate. Km is equal to the substrate concentration at which the reaction velocity is ½ Vmax Km does not vary with enzyme concentration From: Harvey. Biochemistry CONCLUSIONS FROM MICHAELIS-MENTEN EQUATION: Michaelis Menten kinetics Enzyme 1 has a high affinity for its substrate because a low concentration of substrate is needed to half-saturate the enzyme  this is represented in the small Km Enzyme 2 shows a low affinity to the substrate  represented in the large Km CONCLUSIONS FROM MICHAELIS-MENTEN EQUATION: Michaelis Menten kinetics Relationship of velocity V0 to enzyme concentration: → rate of the reaction is directly proportional to the enzyme concentration at all substrate concentrations. (if ½ [E] → ½ Vo and Vmax) Order of reaction: First order [S] < < Km the velocity of the reaction is nearly proportional to the substrate concentration. Zero order [S] > > Km the velocity of the reaction is constant and equal to Vmax  rate of reaction is independent of substrate concentration Michaelis Menten kinetics show hyperbolic curve Michaelis Menten kinetics (i.e.; myoglobin binding to O2 “single substrate”) Allosteric enzymes do not show MichaelisMenten kinetics; they show a sigmoidal curve (i.e.; hemoglobin binding to O2) From: Harvey. Biochemistry Oxyhemoglobin dissociation curve Saturation FYI Rather than relying on a single respiratory protein, many organisms, including teleost, express multiple oxygen carriers with different oxygen-binding properties in order to meet oxygen demand under changing environmental conditions or metabolic challenges. Point mutation → change subunits oxygen affinity → affecting the kinetics parameters of oxygen binding. The Oxygen Transport System in Three Species of the Boreal Fish Family Gadidae: MOLECULAR PHYLOGENY OF HEMOGLOBIN* THE LINEWEAVER-BURK PLOT When V0 is plotted against [S], it is not always possible to determine when Vmax is achieved because of the hyperbolic curve. Enzymes Part 2 Mathematically, the reciprocal 1/v0 and 1/[S] will be plotted to obtain a straight line • Calculation of Km and Vmax → enzyme activity • It can also help to determine the mechanism of action of enzyme inhibitors Hans Lineweaver (1907-2009) and Dean Burk (1904-1988) From: Harvey. Biochemistry FACTORS AFFECTING REACTION VELOCITY Enzymes Part 2 In vitro studies → information on how Substrate concentration: enzymes function in living cells (in vivo) • Maximal velocity  substrate molecules converted to product per unit time (μmol/min) Different enzymes show different responses to changes in  substrate concentration • The rate of an enzyme/catalyzed reaction increases with the substrate  temperature concentration until a maximal velocity  pH • (Vmax) is reached  saturation (substrate are bound to all available binding sites in the enzyme) From: Harvey. Biochemistry Temperature: • Reaction velocity increases with temperature until a peak is reached • Increase is the result of increased number of molecules having sufficient energy to pass over the energy activation barrier • Further elevation of the temperature results in a decrease in reaction velocity, denaturation of proteins  The optimal temperature for most mammalian enzymes is 35-40°C (95-104 °F) Factors affecting reaction velocity Factors affecting reaction velocity pH: • Extreme pH (concentration of H+) conditions can affect the reaction velocity • The pH can affect the ionization state of the active site • Extreme pH conditions can also denature enzymes (the structure of the catalytically active enzyme depends on the ionic character of the amino acid side chains) • pH optimum of enzymes may vary (pepsin, trypsin, alkaline phosphatase) INHIBITION OF ENZYME ACTIVITY Enzymes Part 2 Inhibitor: any substance that can diminish the velocity of an enzyme-catalyzed reaction • Irreversible (covalent bonds; inactivate enzymes, e.g. lead  ferrochelatase) • Reversible inhibitors bind to the enzyme through non-covalent bonds The dilution of the enzyme-inhibitor complex leads to the separation of the reversibly bound inhibitor, enzyme activity can be restored  Competitive inhibition  Noncompetitive inhibition Enzyme function and inhibition https://www.youtube.com/watch?v=PILzvT3spCQ COMPETITIVE INHIBITOR Inhibition of enzyme activity Binds reversibly to the same site that the substrate would normally attach to, competing with substrate for that site. Competitive Inhibitors 1. Effect on Vmax: inhibitor effect is reversed by increasing [S] High enough [S] → reaction velocity reaches the same Vmax observed in absence of inhibitor 2. Effect on Km: competitive inhibitor ↑ Km for given substrate  competitive inhibitor reduces affinity of E for S (competing!!) More substrate is needed to reach ½ Vmax 3. Effect on the Lineweaver-Burk plot 1. Vmax = unchanged 2. Km is higher in the presence of the competitive inhibitor STATIN DRUGS AS EXAMPLES OF COMPETITIVE INHIBITORS Competitive Inhibition Statins (atorvastatin and pravastatin) are structural analogs to this enzyme’s natural substrate Cholesterol biosynthesis hydroxymethylglutaryl-CoA reductase (HMG-CoA reductase) • prevent de novo cholesterol synthesis • help lower plasma cholesterol levels From: Harvey. Biochemistry Enzymes Part 2 NONCOMPETITIVE INHIBITOR • Binds at a site different from the substrate (allosteric site) • It can bind the free enzyme or the ES complex  preventing the reaction from occurring • Conformational change in enzyme or active site of the enzyme → inhibition From: Harvey. Biochemistry Noncompetitive inhibition NONCOMPETITIVE INHIBITION From: Harvey. Biochemistry • Decrease the Vmax of the reaction (↑ [S] does not reverse it – why?) • Km remains unchanged as noncompetitive inhibitors do not interfere with the binding of substrate to the enzyme • These effects can be readily seen when plotting these in a Lineweaver-Burk plot Enzymes Part 2 ENZYME INHIBITORS AS DRUGS At least half of ten most prescribed drugs in the U.S. are enzyme inhibitors • β-lactam antibiotics, such as penicillin and amoxicillin: act by inhibiting enzymes that are important for bacterial cell wall synthesis • Angiotensin-converting enzyme (ACE) inhibitors: block the enzyme that cleaves angiotensin I to the potent vasoconstrictor angiotensin II  cause vasodilation  lower blood pressure • Aspirin: irreversibly inhibits prostaglandin and thromboxane synthesis Enzymes Part 2 HAPPY STUDYING Clara Camargo, DVM [email protected] ©2021 Ross University School of Veterinary Medicine. All rights reserved.