Biochemistry Lecture 3 PDF

The Active Sites of Enzymes LECTURE 3-ENZYMES Enzymes are Remarkable Catalysts -Enzymes typically have two main sites: the catalytic site and the binding site. The catalytic -They speed up important biochemical s...

The Active Sites of Enzymes LECTURE 3-ENZYMES Enzymes are Remarkable Catalysts -Enzymes typically have two main sites: the catalytic site and the binding site. The catalytic -They speed up important biochemical site is where the actual chemical reaction reactions. occurs, and the enzyme facilitates the conversion of substrate(s) into product(s). The -Most enzymes are proteins. Some RNAs are binding site, often called the active site, is enzymes. where the substrate binds to the enzyme, creating an enzyme-substrate complex. This -Enzymes stabilize the transition state, the binding is highly specific, allowing the enzyme highest-energy species in the reaction pathway. to recognize and interact selectively with its substrate, promoting the catalytic reaction. -work in specific temp and ph Enzymes Catalyze Highly Specific Reactions -Reactants in enzyme-catalyzed reactions are called substrates. -Enzymes tend to be highly specific. Proteolytic enzymes for example catalyze the hydrolysis of FIGURE 8.8 Lock-and-key model of peptide bonds between amino acids. enzyme–substrate binding. In this model, the active site of the unbound enzyme is Proteases Readily Break Peptide Bonds complementary in shape to the substrate. Many Enzymes Require Cofactors for Activity -Enzyme cofactors are non-protein molecules or FIGURE 8.9 Induced-fit model of ions that assist enzymes in carrying out enzyme–substrate binding. In this model, the catalysis, which is the acceleration of a chemical enzyme changes shape on substrate binding. reaction. Enzymes and their cofactors work The active site forms a shape complementary to together to lower the activation energy of a the substrate only after the substrate has been chemical reaction, allowing it to proceed more bound. rapidly. The presence of cofactors can be essential for the proper functioning of many enzymes, and their absence can lead to B. THERMODYNAMICS decreased enzymatic activity and, in some Gibbs Free Energy (G) Is a Useful cases, enzyme malfunction. Thermodynamic Function for Understanding Enzymes - The two main classes of cofactors are coenzymes and metals. -An enzyme with its cofactor is called a holoenzyme. Without the cofactor, the enzyme is called an apoenzyme.  -If DG < 0 then the reaction will proceed to the left [C] and [D] will decrease until DG = 0 -.The ΔG of a reaction depends only on the free energy difference between reactants and ea=act eng products and is independent of how the reaction occurs. Energy barrier=transition state= high-energy intermediate state where reactants are in the -.The ΔG of a reaction provides no information process of being converted into products, and about the rate of the reaction. enzymes facilitate the reaction by stabilizing this The Standard Free-energy Change of a Reaction state, lowering the activation energy required is Related to the Equilibrium Constant for the reaction to occur. -For the reaction Enzymes accelerate the Reaction Rate the free energy change is given by but do not change equilibrium where ΔGo is the standard free-energy change, R is the gas constant, T is 298 kelvins, and the square brackets denote concentration in molar. -At equilibrium, ΔG =0; so for the reaction in question FIGURE 8.2 Enzymes accelerate the reaction rate. The same equilibrium point is reached but much more quickly in the presence of an enzyme. Note;product is the same Order reactions -If DG < 0 then the reaction will proceed to the right [C] and [D] will increase until DG = 0 -Reactions that are directly proportional to the reactant concentration are called first-order reactions. First-order rate constants have the units of s−1. -The study of enzyme kinetics is typically the Difference; constant used for first order is 1, the most math intensive component of constant usedfor 2nd order is 2. biochemistry and one of the most daunting aspects of the subject for many students. Michaelis Menten Although attempts are made to simplify the mathematical considerations, sometimes they only serve to confuse or frustrate students. Such is the case with modified enzyme plots, such a Lineweaver-Burk (Figure 4.9.1). Indeed, when Km is [S] at which v = ½ vmax presented by professors as simply another thing kcat = k2 is turnover number of the enzyme to memorize, who can blame students. In kcat/Km is catalytic efficiency of the enzyme reality, both of these plots are aimed at simplifying the determination of parameters, If v is vmax then all enzymes are in [ES] such as 𝐾𝑀 and 𝑉𝑚𝑎𝑥. In making either of these [ES] = [E]T and vmax = k2[E]T modified plots, it is important to recognize that If E is a Michaelis Menten Enzyme then the same data is used as in making a 𝑉 vs. [𝑆] the kinetics are described by plot. The data are simply manipulated to make the plotting easier. Km = Michaelis Menten constant (M) Vmax = maximal reaction rate (M.s-1)  C. ALLOSTERIC REGULATION -Allosteric regulation, broadly speaking, is just any form of regulation where the regulatory molecule (an activator or inhibitor) binds to an enzyme someplace other than the active site. The place where the regulator binds is called the allosteric site. -Pretty much all cases of noncompetitive inhibition and competitive inhibition are forms of allosteric regulation. -However, some enzymes that are allosterically -For a Lineweaver-Burk, the manipulation is regulated have a set of unique properties that using the reciprocal of the values of both the set them apart. These enzymes, which include velocity and the substrate concentration. The some of our key metabolic regulators, are often inverted values are then plotted on a graph as given the name of allosteric enzymes. 1/𝑉 vs. 1/[𝑆]. Because of these inversions, Lineweaver-Burk plots are commonly referred -Allosteric enzymes typically have multiple to as ‘double-reciprocal’ plots. As can be seen at active sites located on different protein left, the value of 𝐾𝑀 on a Lineweaver Burk plot subunits. When an allosteric inhibitor binds to is easily determined as the negative reciprocal an enzyme, all active sites on the protein of the x-intercept, whereas the 𝑉𝑚𝑎𝑥 is the subunits are changed slightly so that they work inverse of the y-intercept. Other related less well. manipulation of kinetic data include Eadie-Hofstee diagrams, which plot V vs V/[S] and give 𝑉𝑚𝑎x -There are also allosteric activators. Some as the Y-axis intercept with the slope of the line allosteric activators bind to locations on an being −𝐾𝑀.. enzyme other than the active site, causing an increase in the function of the active site. Also, Examples of Enzymes that Obey in a process called cooperativity, the substrate Michaelis–Menten Kinetics itself can serve as an allosteric activator: when it -Chymotrypsin (Digestive Tract) binds to one active site, the activity of the other active sites goes up. -Hexokinase I (All Tissues) -Factor IXa (Blood Coagulation) - This is considered allosteric regulation because the substrate affects active sites far from its -Factor Xa (Blood Coagulation) binding site. Examples of Enzymes that do not obey Competitive Inhibition: Michaelis–Menten Kinetics ​-Phosphofructokinase I (All Tissues) -Glycogen phosphorylase (Liver, Muscle)  -Explanation: In noncompetitive inhibition, the inhibitor binds to a site on the enzyme that is not the active site. This binding site, known as the allosteric site, is separate from the active site where the substrate normally binds. The inhibitor and the substrate can both be bound to the enzyme simultaneously, but the inhibitor doesn't compete directly with the substrate for the active site. -Effect: decrease the overall efficiency of the -Explanation: Imagine enzymes as workers enzyme. trying to build a product. In competitive inhibition, a molecule (called the inhibitor) is similar in structure to the substrate (the normal molecule that the enzyme acts on). This inhibitor competes with the substrate for binding to the active site of the enzyme, the place where the reaction occurs. -Effect: the reaction is slowed down or prevented. Allosteric Enzymes Do Not Obey Michaelis–Menten Kinetics FIGURE 8.18 Competitive inhibition illustrated on a double-reciprocal plot. A double-reciprocal -Not all enzymes display Michaelis-Menten plot of enzyme kinetics in the presence and kinetics. absence of a competitive inhibitor illustrates that the inhibitor has no effect on Vmax but q-An important group of enzymes are allosteric increases KM. enzymes, which display cooperative substrate binding. Their kinetics can often be observed as a sigmoidal reaction velocity curve. Noncompetitive Inhibition:  enzyme rate (velocity) is reduced for all values of [S], including Vmax and one-half Vmax but Km remains unchanged because the active site of those enzyme molecules that have not been inhibited is unchanged. FIGURE 8.13 Kinetics for an allosteric enzyme. Allosteric enzymes display a sigmoidal dependence of reaction velocity on substrate concentration. Michealis menten eqn with… This Lineweaver-Burk plot displays these results D. PROTEASES The next figure below is a schematic of the serine protease active site with a peptide substrate bound. Hydrogen atoms are included. The mechanism suggested by the catalytic triad has the following key features: Competitive Inhibition (i) His57 is unprotonated in the resting state of the enzyme. In the presence of a competitive inhibitor, it takes a higher substrate concentration to (ii) His57 is prepared to accept H+ from Ser195 achieve the same velocities that were reached as it attacks the carbonyl carbon of the in its absence. So while Vmax can still be reached dissociated peptide bond. if sufficient substrate is available, one-half Vmax requires a higher [S] than before and thus Km is larger. Noncompetitive Inhibition With noncompetitive inhibition, enzyme molecules that have been bound by the (iii) The role of Asp102 is three-fold: (i) it helps inhibitor are taken out of the game so anchor His57 in its correct conformation, (ii) favors the formation of the neutral form of His57 with the proton on the δ1 N on the imidazole ring, and (iii) stabilization of the substrate (first product) is released as depicted positive charge accumulating on His57 in the in (a), water (the second substrate) is bound in transition state as the Ser195 Oγ atom forms a the active site. (b) A proton is transferred from covalent bond with the carbonyl carbon of the water to His57, leading to the formation of the P1 residue of the substrate. second tetrahedral intermediate. (iv) The "oxyanion hole" - two main-chain amide groups, one from Ser195 itself, the other from residue Gly193, stabilizes a presumed tetrahedral intermediate by favorable dipole-charge interactions. This could be manifested as two strong H-bonds. The deacylation is the reverse of acylation. A The above figure is a representation of the water molecule, taking the place of the released transition state between the initial attack of C-terminal fragment, attacks the carbonyl Ser195 on the carbonyl carbon of the substrate carbon of the acyl enzyme, again assisted by the and the tetrahedral intermediate. The latter, histidine residue in its basic form. illustrated in the figure below, is marked by the full development of a bond between the Oγ atom of Ser195 and the carbonyl carbon of the substrate. The catalytic triad functions as a charge relay system, as indicated by the distribution of partial charges. His57 then donates its extra proton to the γ oxygen of Ser195 as it leaves the substrate acyl carbon, forming the second product and completing the peptide bond hydrolysis. Upon release of the P1 fragment from the enzyme active site, a new peptide substrate can bind to the enzyme, and the hydrolytic mechanism can be reiterated. (v) The transient, protonated form of His57 is thought to transfer its proton to the amine E. VITAMIN K leaving group Vitamin K plays a crucial role in blood clotting by (vi) The form of the enzyme in which Ser195 is facilitating the synthesis of proteins involved in covalently attached to the N-terminal fragment the coagulation Process. of the cleaved substrate by an ester linkage is termed the acyl enzyme. Once the acyl enzyme Vitamin K-dependent coagulation is formed, and the C-terminal fragment of the factors FIX, FVII, FX, and FII refer to coagulation factors in the blood that play a crucial role in the blood clotting process. These factors are initially inactive zymogens(type of enzymes) and require activation by proteolysis to become functional Activation of these factors through proteolysis is a critical step in the coagulation cascade, leading to the formation of a stable blood clot. This cascade is tightly regulated to prevent excessive clotting while maintaining the ability to stop bleeding when necessary. The activation of the zymogens is catalysed by activated Enzymatic cascades are often employed in biochemical systems to achieve a rapid response. In a cascade, an initial signal institutes a series of steps, each of which is catalyzed by Two means of initiating blood clotting have an enzyme. At each step, the signal is amplified. been described, the intrinsic pathway and the For instance, if a signal molecule activates an extrinsic pathway. The intrinsic clotting enzyme that in turn activates 10 enzymes and pathway is activated by exposure of anionic each of the 10 enzymes in turn activates 10 surfaces upon rupture of the endothelial lining additional enzymes, after four steps the original of the blood vessels. The extrinsic pathway, signal will have been amplified 10,000-fold. which appears to be most crucial in blood Hemostasis, the process of blood clot formation clotting, is initiated when trauma exposes tissue and dissolution, requires a cascade of zymogen factor (TF), an integral membrane glycoprotein. activations: the activated form of one clotting Upon exposure to the blood, tissue factor binds factor catalyzes the activation of the next to factor VII to activate factor X. Both the (Figure 10.23). Thus, very small amounts of the intrinsic and extrinsic pathways lead to the initial factors suffice to trigger the cascade, activation of factor X (a serine protease), which ensuring a rapid response to trauma. in turn converts prothrombin into thrombin, the key protease in clotting. Thrombin then amplifies the clotting process by activating enzymes and factors that lead to the generation of yet more thrombin, an example of positive feedback. Note that the active forms of the clotting factors are designated with a subscript “a,” whereas factors that are enzymes or enzyme cofactors activated by thrombin are designated with an asterisk. Prothrombin must bind to Ca2+ to be converted to thrombin Thrombin is synthesized as a zymogen called prothrombin. The inactive molecule is comprised of four major domains, with the serine protease domain at its carboxyl terminus (Figure 10.24). The first domain, called the gla domain, is rich in γ -carboxyglutamate residues (abbreviation gla), and the second and third domains are called kringle domains (named after a Danish pastry that they resemble). These three domains work in concert to keep prothrombin in an inactive form. Moreover, because it is rich in γ -carboxyglutamate, the gla domain is able to bind Ca2+ (Figure 10.25). What is the effect of this binding? The binding of Ca2+ by prothrombin anchors the zymogen to phospholipid membranes derived from blood platelets after injury. This binding is crucial because it brings prothrombin into close proximity to two clotting proteins, factor Xa and Upon thrombin cleavage, amino acid sequences factor Va (a stimulatory protein), that catalyze are exposed in the central globular unit that its conversion into thrombin. Factor Xa cleaves interact with the γ and β subunits of other the bond between arginine 274 and threonine monomers. Polymerization occurs as more fibrin 275 to release a fragment containing the first monomers interact with one another (Figure three domains. Factor Xa also cleaves the bond 10.27). Thus, similar to the activation of between arginine 323 and isoleucine 324 to chymotrypsinogen, peptide-bond cleavage yield active thrombin. exposes new amino termini that can participate in specific interactions. The newly formed “soft clot” is stabilized by the formation of amide Fibrinogen is converted by thrombin bonds between the side chains of lysine and into a fibrin clot glutamine residues in different monomers. Fibrinogen is a large glycoprotein composed of three nonidential chains, Aα, Bβ, and γ and is found in the blood plasma of all vertebrates. Thrombin cleaves four arginine–glycine peptide bonds in the central globular region of fibrinogen. On cleavage, an A peptide of 18 residues is released from each of the two Aα chains, as is a B peptide of 20 residues from each of the two Bβ chains. These A and B peptides are called fibrinopeptides (Figure 10.27). A fibrinogen molecule devoid of these Vitamin K plays a crucial role in blood clotting by fibrinopeptides is called a fibrin monomer and facilitating the synthesis of proteins involved in has the subunit structure (αβγ)2. the coagulation Process. -Vitamin K is converted to a dihydro derivative by y-glutamyl carboxylase, which converts the first 10 glutamate residues in prothrombin into y-carboxyglutamate (Figure 10.29). -γ -carboxyglutamate, a strong chelator of Ca2+, is required for the activation of prothrombin which helps blood to clot. Scott syndrome is a bleeding disorder Scott syndrome is a rare bleeding disorder associated with the maintenance of the asymmetry of the lipid bilayer in the membranes of blood cells, including platelets, leading to reduced thrombin generation and defective wound healing. The asymmetric phospholipid distribution in plasma membranes is normally maintained by energy-dependent Hemophilia A is a bleeding lipid transporters that translocate different disorder phospholipids from one monolayer to the other against their respective concentration gradients. Classic hemophilia, or hemophilia A, is the When cells are activated, or enter apoptosis, best-known clotting defect. This disorder is lipid asymmetry can be disrupted by other lipid genetically transmitted as a sex-linked recessive transporters, which shuttle phospholipids characteristic. In classic hemophilia, factor VIII nonspecifically between the two monolayers. (antihemophilic factor) of the intrinsic pathway This exposes phosphatidylserine (PS) at the is missing or has markedly reduced activity. cells’ outer surface and in cell-derived Although factor VIII is not itself a protease, it microvesicles, which, by providing a catalytic markedly stimulates the activation of factor X, surface for interacting coagulation factors, the final protease of the intrinsic pathway, by promote thrombin generation factor IXa, a serine protease (Figure 10.33). Thus, activation of the intrinsic pathway is severely impaired in classic hemophilia. F. CLASSIFICATION OF ENZYMES Oxidoreductases Leo Ger: Loosing electrons is oxidation G. REGULATION OF ENZYME Gaining electrons is reduction Oxidizing agent is an electron acceptor ACTIVITY Reducing agent is an electron donor Concentration of enzyme and substrate At a constant concentration of enzyme, the Transferases reaction rate increases with increasing substrate concentration until a maximal velocity is reached (Figure 8.4). In contrast, uncatalyzed reactions do not show this saturation effect. The fact that an enzyme-catalyzed reaction has a Kinases transfer a phosphate group from ATP to maximal velocity suggests the formation of a proteins discrete ES complex. At a sufficiently high Phosphatases transfer a phosphate group from substrate concentration, all the catalytic sites a protein to ADP are filled, or saturated, and so the reaction rate cannot increase. Although indirect, the ability to saturate an enzyme with substrate is the most Hydrolases general evidence for the existence of ES complexes. Proteases hydrolyze a peptide bond Lyases Decarboxylases add or remove a carboxyl group and add or release CO2 in biological systems [S] is around Km. Why? Isomerases Covalent modifications The reversible covalent attachment of a chemical group to an amino acid. Alanine racemase converts L-alanine into D-alanine -Recognition of the Target Amino Acid Residue: ligases ->Specific enzymes recognize and target particular amino acid residues within a protein for modification. The choice of the amino acid DNA ligase joins two DNA fragments by forming often depends on the nature of the a phosphodiester bond modification. between the fragments -Activation of the Chemical Group: ->The chemical group that will be attached to as Protein Turnover and Elimination of the amino acid is often activated by an enzyme. Misfolded or Damaged Proteins. This activation step typically involves making the chemical group more reactive, often through the addition of energy or the transfer of a high-energy chemical bond. -Transfer of the Chemical Group: cofactors ->The activated chemical group is then Cofactors are non-protein chemical compounds transferred to the target amino acid residue in or metallic ions that are essential for the activity the protein. This transfer is typically mediated of some enzymes. They play a crucial role in by enzymes known as transferases. modulating enzymatic activity by facilitating the catalytic function of the enzyme. Cofactors can -Formation of a Covalent Bond: be broadly classified into two types: coenzymes and metal ions. ->The transferred chemical group forms a covalent bond with the amino acid residue, resulting in the modification of the protein. This covalent attachment is usually reversible, allowing for dynamic regulation of protein function. -Regulation and Reversibility: ->The covalent attachment of the chemical group can be reversed by specific enzymes known as hydrolases or by other regulatory processes. This reversibility is crucial for the dynamic regulation of cellular processes. ex; proteolysis Proteolysis is the biological process of breaking down proteins into smaller fragments, peptides, or individual amino acids. This essential cellular mechanism is carried out by enzymes called proteases or peptidases. Proteolysis serves several critical roles in cellular regulation, such