Enzymes: Lecture Notes PDF

LECTURE 3-ENZYMES Enzymes are Remarkable Catalysts -They speed up important biochemical reactions. -Most enzymes are proteins. Some RNAs are enzymes. -Enzymes stabilize the transition state, the highest-energy species in the reaction pathway. -work in specific temp and ph Enzymes Catalyze Hi...

LECTURE 3-ENZYMES Enzymes are Remarkable Catalysts -They speed up important biochemical reactions. -Most enzymes are proteins. Some RNAs are enzymes. -Enzymes stabilize the transition state, the highest-energy species in the reaction pathway. -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 peptide bonds between amino acids. ​ Proteases Readily Break Peptide Bonds ​ Many Enzymes Require Cofactors for Activity -Enzyme cofactors are non-protein molecules or ions that assist enzymes in carrying out  catalysis, which is the acceleration of a chemical reaction. Enzymes and their cofactors work together to lower the activation energy of a chemical reaction, allowing it to proceed more rapidly. The presence of cofactors can be essential for the proper functioning of many enzymes, and their absence can lead to decreased enzymatic activity and, in some cases, enzyme malfunction. - 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. The Active Sites of Enzymes -Enzymes typically have two main sites: the catalytic site and the binding site. The catalytic site is where the actual chemical reaction occurs, and the enzyme facilitates the conversion of substrate(s) into product(s). The binding site, often called the active site, is where the substrate binds to the enzyme, creating an enzyme-substrate complex. This binding is highly specific, allowing the enzyme to recognize and interact selectively with its substrate, promoting the catalytic reaction. FIGURE 8.8 Lock-and-key model of enzyme–substrate binding. In this model, the active site of the unbound enzyme is complementary in shape to the substrate. FIGURE 8.9 Induced-fit model of enzyme–substrate binding. In this model, the enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound. B. THERMODYNAMICS Gibbs Free Energy (G) Is a Useful Thermodynamic Function for Understanding Enzymes -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 products and is independent of how the reaction occurs. -.The ΔG of a reaction provides no information about the rate of the reaction. The Standard Free-energy Change of a Reaction is Related to the Equilibrium Constant -For the reaction the free energy change is given by 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 ea=act eng Energy barrier=transition state= high-energy intermediate state where reactants are in the process of being converted into products, and enzymes facilitate the reaction by stabilizing this state, lowering the activation energy required for the reaction to occur. Enzymes accelerate the Reaction Rate but do not change equilibrium 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. Difference; constant used for first order is 1, the constant usedfor 2nd order is 2. Michaelis Menten Km is[S]atwhichv=1⁄2vmax kcat = k2 is turnover number of the enzyme kcat/Km is catalytic efficiency of the enzyme If v is vmax then all enzymes are in [ES] [ES] = [E]T and vmax = k2[E]T If E is a Michaelis Menten Enzyme then the kinetics are described by Km = Michaelis Menten constant (M) Vmax = maximal reaction rate (M.s-1) -The study of enzyme kinetics is typically the most math intensive component of biochemistry and one of the most daunting aspects of the subject for many students. 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 presented by professors as simply another thing to memorize, who can blame students. In reality, both of these plots are aimed at simplifying the determination of parameters, such as 𝐾𝑀 and 𝑉𝑚𝑎𝑥. In making either of these modified plots, it is important to recognize that the same data is used as in making a 𝑉 vs. [𝑆] plot. The data are simply manipulated to make the plotting easier. -For a Lineweaver-Burk, the manipulation is using the reciprocal of the values of both the velocity and the substrate concentration. The inverted values are then plotted on a graph as 1/𝑉 vs. 1/[𝑆]. Because of these inversions, Lineweaver-Burk plots are commonly referred to as ‘double-reciprocal’ plots. As can be seen at left, the value of 𝐾𝑀 on a Lineweaver Burk plot is easily determined as the negative reciprocal of the x-intercept, whereas the 𝑉𝑚𝑎𝑥 is the inverse of the y-intercept. Other related manipulation of kinetic data include Eadie-Hofstee diagrams, which plot V vs V/[S] and give 𝑉𝑚𝑎x as the Y-axis intercept with the slope of the line being −𝐾𝑀.. Examples of Enzymes that Obey Michaelis–Menten Kinetics -Chymotrypsin (Digestive Tract) 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 regulated have a set of unique properties that set them apart. These enzymes, which include some of our key metabolic regulators, are often given the name of allosteric enzymes. -Allosteric enzymes typically have multiple active sites located on different protein subunits. When an allosteric inhibitor binds to an enzyme, all active sites on the protein subunits are changed slightly so that they work less well. -There are also allosteric activators. Some allosteric activators bind to locations on an enzyme other than the active site, causing an increase in the function of the active site. Also, in a process called cooperativity, the substrate itself can serve as an allosteric activator: when it binds to one active site, the activity of the other active sites goes up. - This is considered allosteric regulation because the substrate affects active sites far from its binding site. Competitive Inhibition: -Hexokinase I -Factor IXa -Factor Xa (All Tissues) (Blood Coagulation) (Blood Coagulation)  Examples of Enzymes that do not obey 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 enzyme. -Explanation: Imagine enzymes as workers 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. FIGURE 8.18 Competitive inhibition illustrated on a double-reciprocal plot. A double-reciprocal plot of enzyme kinetics in the presence and absence of a competitive inhibitor illustrates that the inhibitor has no effect on Vmax but increases KM. Noncompetitive Inhibition: Allosteric Enzymes Do Not Obey Michaelis–Menten Kinetics -Not all enzymes display Michaelis-Menten kinetics. q-An important group of enzymes are allosteric enzymes, which display cooperative substrate binding. Their kinetics can often be observed as a sigmoidal reaction velocity curve. FIGURE 8.13 Kinetics for an allosteric enzyme. Allosteric enzymes display a sigmoidal dependence of reaction velocity on substrate concentration.  Michealis menten eqn with... Competitive Inhibition In the presence of a competitive inhibitor, it takes a higher substrate concentration to achieve the same velocities that were reached in its absence. So while Vmax can still be reached 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 inhibitor are taken out of the game so 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: (i) His57 is unprotonated in the resting state of the enzyme. (ii) His57 is prepared to accept H+ from Ser195 as it attacks the carbonyl carbon of the dissociated peptide bond. (iii) The role of Asp102 is three-fold: (i) it helps 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 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. imidazole ring, and (iii) stabilization of the positive charge accumulating on His57 in the transition state as the Ser195 Oγ atom forms a covalent bond with the carbonyl carbon of the P1 residue of the substrate. (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 above figure is a representation of the transition state between the initial attack of Ser195 on the carbonyl carbon of the substrate and the tetrahedral intermediate. The latter, 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. substrate (first product) is released as depicted in (a), water (the second substrate) is bound in the active site. (b) A proton is transferred from water to His57, leading to the formation of the second tetrahedral intermediate. The deacylation is the reverse of acylation. A water molecule, taking the place of the released C-terminal fragment, attacks the carbonyl carbon of the acyl enzyme, again assisted by the histidine residue in its basic form. 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.