Enzyme Kinetics


Enzyme Kinetics

      • General properties of enzymes
        • At the active sites of enzymes, substrates (reactants) bind ‘‘tightly’’ to the enzyme, and the enzyme then catalyzes their conversion to products, which are released.
        • The transition state is a high-energy reactive conformation of reactants with enzyme
        • The transition state has a very high probability of a structural rearrangement of bondsproducing the products of the reaction.
        • Enzymes are highly specificfor their substrates and products.
          • Many enzymes recognize only a single compound as a substrate.
          • proteases that hydrolyze proteins to peptides are less specific.
        • Enzymes as drug targets account for about 30% of pharmacotherapeutic agents
        • Many enzymes require small organic molecules, or cofactors (often called coenzymes), to catalyze their reactions.
          • The cofactors are frequently derivatives of vitamins or metal ions.
        • Enzymes decrease the energy of activation (Ea) for a reaction and hence speed up the rate of reactions.
          • Enzymes do not affect the thermodynamics (DG) of the reaction (net free energy change for the reaction or equilibrium concentrations of the substrates and products).(MCQ)
          • ThethermodynamicsofthereactionremainUNCHANGED
          • The transition state is at the apex (the top) of the energy diagram between reactants andproducts.(MCQ)
          • The difference in the average free energy of the reactants and the average free energy of thetransition state is the activation energy barrier (free energy of activation; Ea).(MCQ)
        • Dependence of velocity on enzyme and substrate concentrations, temperature, and pH
          • The velocity of a reaction, v, increases with the enzyme concentration, [E], if the substrate con- centration, [S], is constant.
          • If [E] is constant, v increases with [S] until the maximum velocity, Vmaxis attained.(MCQ)
          • AtVmax,alltheactivesitesoftheenzymearesaturatedwithsubstrate.(MCQ)
          • The velocity of a reaction increases with temperature until a maximum (37° C in humans) is reached, after which the velocity decreases owing to denaturation of the enzyme (MCQ)
        • Each enzyme-catalyzed reaction has an optimal pH(not always physiologic pH).
          • The optimal pH is the pH at which the enzyme and substrate exhibit the most efficient interaction and the velocity is at a maximum.
          • Changes in the pH can alter the interaction between enzyme and substrate such that the reac- tion proceeds at a slower rate.
          • If the pH is too high or too low, the enzyme can also undergo denaturation(MCQ)
        • The michaelis-menten equation
          • If, during a reaction, an enzyme–substrate complexis formed that dissociates (becoming free enzyme and substrate) orreacts(to release the product and regenerate the free enzyme), then:
          • 1
                • where Km =(k2 + k3)/k1
                • Vmax is the maximum velocity, or how fast the enzyme can go at full ‘‘speed.’’
                  • Vmax is reached when all of the enzyme is in the enzyme–substrate complex.(MCQ)
                • Km is the substrate concentration at which v = 1/ 2 Vmax.(MCQ)
                  • Km approximately describes the affinity of the substrate for the enzyme.
                  • The lower the value ofKm, the higher the apparent affinity for substrate.(MCQ)
                  • When [S] =Km, the Michaelis-Menten equation yields v = 1/2 Vmax.
              • When the velocity is plotted versus [S], a hyperbolic curve is produced (MCQ)
                • At low substrate concentration (left part of the curve, below Km), the reaction rate increases sharply with increasing substrate concentration because there is abundant free enzyme available (E) to bind added substrate.
                • At high substrate concentration, the reaction rate reaches a plateau (Vmax) as the enzymeactive sites are saturated with substrate (ES complex), and there is no free enzyme to bind the added substrate.
              • The Line weaver-Burk plot(MCQ)
                • Because of the difficulty of exactly determining Vmax from a hyperbolic curve, the Michaelis- Menten equation was transformed by Lineweaver and Burk into an equation for a straight line.
              • This is a double reciprocal plot of 1/V versus 1/[S].(MCQ)
            • Inhibitors
              • Competitive inhibitors (MCQ)
                • compete with the substrate for binding at the active site of the enzyme and form an enzyme–inhibitor complex, EI, with the free enzyme only
                • Structurally, these inhibitors ar(MCQ)e similar to substrate because they compete for the same site.
                • Competitive inhibition is reversed by increasing substrate [S].
                • Vmax remains the same, but the apparent Km (K’m) is increased.(MCQ)
                • For Lineweaver-Burk plots, lines for the inhibited reaction intersect on the Y-axis with thosefor the uninhibited reaction.
              • Noncompetitive inhibitors (MCQ)
                • bind to the enzyme or the enzyme–substrate complex at a site distinct from the active site, decreasing the activity of the
                • Vmax is decreased.(MCQ)
                • Inhibition cannot be overcome by increasing substrate.(MCQ)
                • Structurally, these inhibitors are not similar to substrate.
              • Irreversible inhibitors
                  • enzymeinactivators(MCQ)
                  • bind covalently to the enzyme and inactivate it.
                  • Their kinetics appear exactly like noncompetitive inhibition
                  • anincrease in inhibition with length of exposure (MCQ)
                  • inability to remove by dilution (because they are covalently bound).


              • Allosteric enzymes
                • Virtually every metabolic pathway is subject to feedback control, and allosteric enzymes are used. (MCQ)
                • This is a component of feedback inhibition, whereby the concentration of the end product of a pathway is ‘‘monitored’’ to shut off the first (usually an allosteric) enzyme in a pathway to prevent unwanted and wasted production of intermediate compounds.
                • Allosteric enzymes are oligomeric (multiple subunits)(MCQ)
                • activators or inhibitors bindat sites other than (but interacting with) the active substrate binding sites through conformational changes
                • Sigmoidal curves
                  • generated by plots of v versus [S].(MCQ)
                  • Allosteric enzymes do not obey Michaelis- Menten kinetics
                  • An allosteric enzyme has two or more subunits, each with substrate-binding sites that exhibitcooperativity.(MCQ)
                    • Binding of a substrate molecule at one site facilitates binding of other substrate molecules at other sites.
                  • Allosteric activatorscause the enzyme to bind substrate more readily.
                  • Allosteric inhibitorscause the enzyme to bind substrate less readily.
                  • Similar effects occur during O2 binding to hemoglobin
              • Phosphorylation affects many enzymes.
                • Pyruvatedehydrogenaseandglycogensynthaseareinhibitedbyphosphorylation(MCQ)
                • glycogenphosphorylase is activated by phosphorylation(MCQ)
                • Phosphatases that remove phosphate groups alter the activities of these enzymes.
              • Regulation by protein–protein interactions
                • Proteins can bind to enzymes, altering their activity.
                • For example, regulatory subunits inhibit the activity of protein kinase A
                • When these regulatory subunits bind cAMP and are released from the enzyme, the catalytic subunits become active.(MCQ)
                • Enzymes can be arranged as enzyme cascades, exponentially amplifying the availability/activity of products in the pathway (e.g., hormone activation, blood clotting).
              • Isoenzymes
                • Isoenzymes (or isozymes) are enzymes composed of different amino acid sequences that catalyze the same reaction.
                • Isozymes also differ in many of their physical properties.
                • Creatine kinase  – MB fraction is most prevalent in heart muscle.(MCQ)


              • Applied aspects :
                • Optimal pH
                  • The optimal pH for pepsin is 2, reflecting its need as a digestive enzyme in theacidic gastric juice of the stomach.
                  • The optimal pH for alkaline phosphatase is 9, reflecting the basic pH environment in bone.(MCQ)
                • Isoniazid
                  • used in the treatment of tuberculosis
                  • is acetylated by anN-acetyltransferase.
                  • A polymorphism of the enzyme exists
                  • fastacetylators/metabolizers clear the drug from blood about 300% faster
                  • slowacetylators/poor metabolizers, in whom the presence of drug is prolonged, causing hepatotoxicity and neuropathy. (MCQ)
                  • The Km (affinity of isoniazid substrate) is normal, but the Vmax of ‘‘fast’’ N-acetyltransferase, is three times normal.(MCQ)
                • Hypersensitivity to alcohol
                  • exists when drinking small amounts of alcohol causesfacial flushing and tachycardia
                  • Alcohol dehydrogenase generates acetaldehyde, which is converted to acetate by aldehyde dehydrogenase
                  • aldehyde dehydrogenase existsin two forms, a high-affinity (low Km) form and a low-affinity (high Km) form. (MCQ)
                  • Those sensitive to alcohol lack the high-affinity form, resulting in excess acetaldehyde and, hence, vasodilation.
                • Physostigmine,
                  • a competitive reversible inhibitor ofacetylcholinesterase
                  • used to treat a variety of diseases such as glaucoma  and myasthenia gravis
                • Angiotensin-converting enzyme (ACE) inhibitors
                  • captopril, enalapril,and lisinopril,
                  • inhibit formation of angiotensin II, an octapeptide from angiotensin I.
                • ethylenediaminetetraacetic acid (EDTA)
                  • A common noncompetitive inhibitor
                  • Causes chelation
                  • result in removal of required divalent metal ions from the active site of enzymes.
                  • Blood of patients is collected in tubes with EDTA to inhibit both calcium-activated proteases and the blood coagulation pathway.(MCQ)
                • Nerve gases- tabun and sarin, andalkylphosphate insecticides (malathion)
                  • irreversible inhibitors of acetylcholinesterase.
                  • These compounds are also termed ‘‘suicide’’ inhibitors, creating a reactive group irreversibly reacting in the active site, forming an extremely stable intermediate.(MCQ)

                  An Introduction to Enzyme Kinetics
                  Let’s take a look at how enzymes work through a mathematical lens.
                  Enzyme kinetics introduction
                  In this video I provide a basic introduction to enzyme kinetics, focusing on an intuitive understanding
                  Enzyme Kinetics
                  Enzyme Kinetics
                  Derivation of the Michaelis-Menten equation.
                  MCAT Biology Lecture: Enzyme Kinetics