Biochemistry I CHM219 Lecture Notes PDF

BIOCHEMISTRY I CHM219 Assist. Prof. Dr. ESRA AYDEMİR  Enzymes II: The Biological Catalysts  Outline: The Kinetics of Enzymatic Catalysis Enzyme Inhibition Cofactors, Vitamins, and Essential Metals The Diversity of Enzymatic Function Nonprotein Biocatalysts: Catalytic Nucleic...

BIOCHEMISTRY I CHM219 Assist. Prof. Dr. ESRA AYDEMİR  Enzymes II: The Biological Catalysts  Outline: The Kinetics of Enzymatic Catalysis Enzyme Inhibition Cofactors, Vitamins, and Essential Metals The Diversity of Enzymatic Function Nonprotein Biocatalysts: Catalytic Nucleic Acids The Regulation of Enzyme Activity: Allosteric Enzymes Covalent Modifications Used to Regulate Enzyme Activity Conformational Selection vs. Induced Fit: Induced fit implies conformational homogeneity in the unbound enzyme, which is then distorted upon substrate binding. Conformational selection implies that the unbound enzyme is present in multiple conformations; but, the substrate can only bind to unbound enzyme that is in the same conformation as that of the ES complex. Substrate binding will perturb the conformational equilibrium of the unbound enzyme, driving more E into the ES conformation by Le Chatelier’s principle.  The Kinetics of Enzymatic Catalysis An expression for a simple reaction involving a single substrate and product was shown as: If we analyze the initial rate of an enzyme-catalyzed reaction (i.e., before a significant concentration of P appears) and we assume that k1, k-1, and k3 >> k2, the equation above simplifies to: Where kcat is the apparent rate constant for the rate-determining conversion of substrate to product.  We have assumed that initial reaction conditions are such that the reverse reaction between E and P is negligible. The catalytic formation of the product, with enzyme regeneration, will then be a simple first-order reaction, and its rate will be determined solely by the concentration of ES and the value of kcat. Therefore, the reaction rate, or velocity, defined as the observed rate of formation of products, can be expressed as v = kcat[ES] In practice, [ES] is difficult to measure in kinetics experiments.  It is desirable to express the rate, v, in terms of the substrate concentration [S] and the total enzyme concentration. This suggests that E and S should be in equilibrium with ES, with an equilibrium dissociation constant This is usually an incorrect assumption, but under certain circumstances this approximation is valid. When the reaction is started by mixing enzymes and substrates, the ES concentration builds up at first, but quickly reaches a steady state, in which it remains almost constant. This steady state will persist until almost all of the substrate has been consumed. Because the steady state accounts for nearly all the reaction time, we can calculate the reaction velocity by assuming steady-state conditions. The Michaelis–Menten equation is the rate equation for a one- substrate enzyme-catalyzed reaction. This equation relates the initial reaction rate (v0), the maximum reaction rate (Vmax), and the initial substrate concentration [S] through the Michaelis constant KM— Km: a measure of the substrate-binding affinity. Reaction velocity as a function of substrate concentration: This graph shows the variation of reaction velocity with substrate concentration according to the Michaelis–Menten model of enzyme kinetics. The values of v plotted here are determined from the initial rates of the reaction. At the point where [S] = KM the reaction has exactly half its maximum velocity. Note that Vmax is approached asymptotically.  The steady-state assumption proposes that the concentration of enzyme–substrate complex remains nearly constant through much of the reaction. The Michaelis constant, KM, indicates the substrate concentration at which the reaction rate is ½ Vmax. The turnover number, kcat, measures the rate of the catalytic process. The ratio kcat/KM is a convenient measure of enzyme efficiency. Analysis of initial rates: a)Several reactions are performed with varying concentrations of substrate and the values of the initial rates are determined from the slopes of the curves in the early phase for each reaction. b)Initial rate data, determined as described in panel A, are plotted as a function of the substrate concentration. The enzyme appears to obey the Michaelis–Menten kinetic model. A Lineweaver–Burk plot: In this double reciprocal plot, 1/v is graphed versus 1/[S]. Note that a linear extrapolation of the data gives both Vmax and KM. Useful for determining important variables in enzyme kinetics, BUT it is prone to error. The y-axis of the plot takes the reciprocal of the rate of reaction, meaning small errors in measurement are more noticeable. Additionally because most points on the plot are found far to the right of the y-axis large values of [S] (and hence small values for 1/[S] on the plot) are often not possible due to limited solubility An Eadie–Hofstee plot: Graphing v versus V/[S], we obtain Vmax at (V/[S]) = 0 and KM from the slope of the line. A graphical representation of enzyme kinetics in which velocity is plotted as a function of the ratio between velocity and substrate concentration  Lineweaver–Burk plots or Eadie–Hofstee plots provide convenient ways to determine KM and kcat from intial-rate data. The observed effects of an amino acid mutation in an enzyme active site on KM and kcat can be used to identify the role of the amino acid in substrate binding (KM effects) and transition-state stabilization (kcat effects).  Multisubstrate reactions fall into several classes, depending on the order of substrate binding: o Random o Ordered o Ping-pong Random Substrate Binding: Either substrate can be bound first, although in many cases one substrate will be favored for initial binding, and its binding may promote the binding of the other. The general pathway is: Ordered Substrate Binding: In some cases, one substrate must bind before a second substrate can bind significantly. This mechanism is often observed in oxidations of substrates by the cofactor NAD+. The Ping-Pong Mechanism: In a “ping-pong” reaction: o One substrate is bound, one product is released. o A second substrate comes in, and a second product is released. Here E* is a modified form of the enzyme, often carrying a fragment of S1.  Enzyme Inhibition Inhibition of enzymes can be either reversible or irreversible. A competitive inhibitor competes with substrate for the enzyme active site. It increases the apparent KM. Both substrate and inhibitor can fit the active site. Substrate can be processed by the enzyme, but inhibitor cannot. Effects of competitive inhibition on enzyme kinetics: The effect of a competitive inhibitor (I) on reaction velocity at different substrate concentrations. Lineweaver–Burk plots of the reactions with and without a competitive inhibitor: The lines cross the 1/v axis at the same Vmax, showing that I is a competitive inhibitor. Determination of KM and KI: If the measurement of is repeated at different concentrations of I, KI can be determined from the slope of the line, and the true KM from the line’s intercept where [I] = 0. A substrate and its competitive inhibitor: The substrate UpA and the structurally similar molecule UpcA are competitors for the enzyme ribonuclease. The single difference between the substrate and the inhibitor is shown in red.  An uncompetitive inhibitor does not compete for the active site but affects the catalytic event. It reduces both the apparent Vmax and apparent KM. These effects cannot be reversed by increasing [S].  An uncompetitive inhibitor does not compete for the active site but affects the catalytic event. Effects of uncompetitive inhibition on enzyme kinetics: (a)The effect of an uncompetitive inhibitor (I) on reaction velocity at different substrate concentrations. (b)Lineweaver–Burk plots of the reactions shown in (a). The lines are parallel and cross 1/v axis at different points, clearly distinguishing this situation from competitive inhibition. A model for mixed inhibition: The inhibitor binds at a site on the enzyme surface different from that of the substrate. In this simplified example, the inhibitor binds to both free enzyme and the ES complex. EI has reduced substrate binding affinity compared to free enzyme. The EIS complex cannot carry out the catalytic event. A model for mixed inhibition: Lineweaver–Burk plot for mixed inhibition kinetics: Vmax is decreased by a factor of and KM is increased. Compare this plot with those previously shown to find the features that distinguish competitive, uncompetitive, and mixed modes of inhibition.  Many irreversible inhibitors bind covalently to the active sites of enzymes. Irreversible inhibition by adduct formation: Diisopropyl fluorophosphate (DFP) reacts with a serine group on a protein to form a covalent adduct. The covalent bond renders the catalytically important serine ineffective in catalysis. The adduct also may block substrate binding to the active site. Cofactors, Vitamins, and Essential Metals Many essential vitamins are constituents of enzyme cofactors.  The nicotinamide nucleotides, a major example being nicotinamide adenine dinucleotide (NAD+) derived from the vitamin niacin.  The nicotinamide portion is the metabolically relevant part of because it is capable of being reduced and thus can serve as an oxidizing agent to which two electrons and a proton are added to the nicotinamide ring: NAD+ + 2e- + H+ → NADH The reaction is reversible (i.e., NADH acts as a reducing agent in several reactions) and is formally a hydride ion transfer: NAD+ + H- → NADH  A typical reaction in which NAD+ acts as an oxidizing agent is the conversion of alcohols to aldehydes or ketones. For example, by the alcohol dehydrogenase of liver.  The Diversity of Enzymatic Function Some enzymes require metal ions for their catalytic function, such as carboxypeptidease A. The zinc ion binds a water molecule and serves as an electrostatic catalyst to promote hydrolysis of the C-terminal amino acid from a peptide substrate. It does so by stabilizing the negative charge on the oxygen in the tetrahedral transition state. A rational naming and numbering system has been devised by the Enzyme Commission of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzymes are divided into six major classes as follows: 1. Oxidoreductases catalyze oxidation–reduction reactions. 2. Transferases catalyze transfer of functional groups from one molecule to another. 3. Hydrolases catalyze hydrolytic cleavage. 4. Lyases catalyze removal of a group from or addition of a group to a double bond, or other cleavages involving electron rearrangement. 5. Isomerases catalyze intramolecular rearrangement. 6. Ligases catalyze reactions in which two molecules are joined. The IUBMB Enzyme Commission (EC) has given each enzyme a number with four parts, such as: EC 3.4.21.5. The first three numbers define major class, subclass, and sub- subclass, respectively.  Nonprotein Biocatalysts: Catalytic Nucleic Acids Ribozymes, a class of ribonucleic acids, also function as biological catalysts. The production of tRNA from pre-tRNA is catalyzed by an RNA–protein complex called ribonuclease P. The RNA portion of ribonuclease P can by itself catalyze the hydrolysis of the specific phosphodiester bond indicated by the red wedge. The Regulation of Enzyme Activity: Allosteric Enzymes Regulation of enzyme activity is essential for the efficient and ordered flow of metabolism. Feedback control is important in the efficient regulation of complex metabolic pathways. Effect of cooperative substrate binding on enzyme kinetics: (a) Comparison of v vs. [S] curves for a noncooperative enzyme and an allosteric enzyme with cooperative binding. The two enzymes are assumed to have the same Vmax. (b) Lineweaver-Burk Plot. The T state has a high KM (Vmax is achieved at higher [S]). As more S is bound, the equilibrium shifts toward the R state, which has a lower KM. Effect of extreme homoallostery: The v vs. [S] curve is shown for a hypothetical enzyme with extreme positive cooperativity in substrate binding. At concentrations below the enzyme is almost inactive; above this concentration, it is very active. Substrate can easily accumulate to the level but at higher concentrations it will be processed rapidly. The vertical blue line represents the homeostatic concentration range for S.  Allosteric enzymes show cooperative substrate binding and can respond to a variety of inhibitors and activators. In the absence of activation or inhibitors, the v vs. [S] curve is sigmoidal. Activators shift the system toward the R state. Inhibitors stabilize the T state. [S]c represents the homeostatic concentration range for S. Note that effectors significantly alter the activity of the enzyme over this range of [S]. Control points in pyrimidine synthesis: The formation of N-carbamoyl-L-aspartate from carbamoyl phosphate and aspartate is the first committed step in a series of reactions that leads to synthesis of pyrimidine nucleotides, so control at or near this point is essential. In prokaryotes, the aspartate carbamoyltransferase (ATCase) is regulated; in most eukaryotes, regulation is on the preceding step, catalyzed by carbamoyl phosphate synthetase II. Regulation of ATCase by ATP and CTP: ATP is an activator. CTP is an inhibitor. The curve marked as “control” shows the behavior of the enzyme in the absence of both regulators. N-Carbamoyl-L-aspartate (CAA) is the product of the reaction. (a) Quaternary structure of ATCase in the T state. The enzyme has the six catalytic subunits (C) and six regulatory subunits (R). Six catalytic sites lie in or near the grooves between the catalytic subunits. Regulatory sites lie on the outer surfaces of the regulatory subunits. (b) Transition of ATCase to the R state. The transition involves a rotation of the regulatory subunits, which pushes the two tiers of catalytic subunits apart and rotates them slightly about the three-fold axis Covalent Modifications Used to Regulate Enzyme Activity Reversible covalent modification by kinases/ phosphatases: The target residues for ATP-dependent phosphorylation by kinases are serine, threonine, or tyrosine. The phosphoprotein is dephosphorylated by a phosphatase-catalyzed hydrolysis reaction.  Some enzymes, such as pancreatic proteases, are irreversibly switched on by proteolytic cleavage. Zymogens are activation by proteolytic cleavage. This schematic view shows the activation of pancreatic zymogens, molecules that become catalytically active when cleaved. Activation of chymotrypsinogen: A series of cleavages produces the enzyme chymotrypsin, with the disulfide bonds continuing to hold the structure together. The initial cleavage between amino acids 15 and 16 (arrow) results in the formation of - chymotrypsin. Subsequent removal of the segments shown in black yields -chymotrypsin.  Blood clotting involves a cascade of proteolytic activation of specific proteases, culminating in the transition of fibrinogen to fibrin. Each factor (protease) in the pathway can exist in an inactive form (red) or an active form (green). The cascade of proteolytic activations can start from exposure of blood at damaged tissue surfaces (intrinsic pathway) or from internal trauma to blood vessels (extrinsic pathway). The common result is activation of fibrinogen to clotting fibrin.  Covalent Modifications Used to Regulate Enzyme Activity Formation of a blood clot: a)Red blood cells enmeshed in the insoluble strands of a fibrin clot. b)Electron micrograph of part of a fibrin fiber. c)Schematic view of how fibrin monomers are thought to associate to form a fiber. Removal of fibrinopeptides A and B from fibrinogen by thrombin makes sites accessible for association with complementary sites a and b on adjacent monomers. The molecules are believed to overlap as shown because the striations seen in the fibers are 23 nm wide, exactly half the length of the fibrinogen molecule.