Biochemistry I CHM219 Fall 2021 PDF

BIOCHEMISTRY I CHM219 Assist. Prof. Dr. ESRA AYDEMİR  Enzymes I: The Biological Catalysts  Outline: The Role of Enzymes Chemical Reaction Rates and the Effects of Catalysts—A Review How Enzymes Act as Catalysts: Principles and Examples The Kinetics of Enzymatic Catalysis E...

BIOCHEMISTRY I CHM219 Assist. Prof. Dr. ESRA AYDEMİR  Enzymes I: The Biological Catalysts  Outline: The Role of Enzymes Chemical Reaction Rates and the Effects of Catalysts—A Review How Enzymes Act as Catalysts: Principles and Examples 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  History of Enzymes Much of the history of biochemistry Biological catalysis was first recognized and described in the late 1700s, in studies on the digestion of meat by secretions of the stomach. Research continued in the 1800s with examinations of the conversion of starch to sugar by saliva and various plant extracts. In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by “ferments.” He postulated that these ferments were inseparable from the structure of living yeast cells. This view, called vitalism, prevailed for decades. Then in 1897, Eduard Buchner discovered that cell-free yeast extracts could ferment sugar to alcohol, proving that fermentation was promoted by molecules that continued to function when removed from cells. Buchner’s experiment marked the end of vitalistic notions and the dawn of the science of biochemistry. Frederick W. Kühne later gave the name enzymes (from the Greek enzymos, “leavened”) to the molecules detected by Buchner  All enzymes were proteins The isolation and crystallization of urease by James Sumner in 1926 was a breakthrough in early enzyme studies. Sumner found that urease crystals consisted entirely of protein, and he postulated that all enzymes were proteins In 1930s Sumner’s conclusion was widely accepted, after John Northrop and Moses Kunitz crystallized pepsin, trypsin, and other digestive enzymes and found them also to be proteins. James Sumner  The Role of Enzymes In general terms, a catalyst is a substance that increases the rate, or velocity, of a chemical reaction without itself being changed in the overall process. The protein trypsin catalyzes hydrolysis of peptide bonds in proteins and polypeptides. The substance that is acted on by an enzyme is called the substrate of that enzyme. Two facts deserve emphasis: 1.Although a true catalyst participates in the reaction process, it is unchanged by the process. For example, after catalyzing the decomposition of an molecule, catalase is found again in exactly the same state as before, ready for another round. In contrast, although hemoglobin accelerates the rate of H2O2 decomposition, it is oxidized in the process, from the active Fe2+ to the inactive Fe3+ form; thus, hemoglobin is not a true catalyst for this reaction. 2.Catalysts change rates of processes but do not affect the position of equilibrium for a reaction. A thermodynamically favorable process is not made more favorable, nor is an unfavorable process made favorable, by the presence of a catalyst. The equilibrium state is just approached more quickly.  Chemical Reaction Rates and the Effects of Catalysts A first-order reaction is one whose rate is directly proportional to the first power of the reactant concentration. A first-order reaction is characterized by single exponential decay of the reactant. Determining the order and rate constant of an irreversible first-order reaction: Graphs (a) and (b) analyze the rate of a single reaction, with time expressed as multiples of the half-life (t1/2) of the reactant. Note that for each interval of t1/2 the reactant concentration is halved. a)A graph of [A] versus t shows that the rate, defined as the slope of the curve, decreases as the reaction continues. b) A graph of ln[A] versus t, when linear, indicates that the reaction follows the equation [A]t = [A]oe-kt and is first-order. The slope of this line (d ln[A]/dt) is equal to - k1.  A first-order rate constant has units of (time)-1, whereas a second-order rate constant has units of (concentration) -1(time) -1. Often, however, the analysis of complex multistep reaction schemes can be simplified by the recognition of a rate-limiting step. The rate-limiting step is the slowest step in a multistep process. As such, it determines the experimentally observed rate for the entire process.  Barriers to chemical reactions occur because a reactant molecule must pass through a high-energy transition state to form products. This free energy barrier is called the activation energy. Catalysts increase reaction rates by lowering the activation energy. Free energy diagrams for the simple reaction A  B: a)Only the free energy difference between the initial state and the final state is revealed. b) Free energy diagram filled in to include the transition state through which the molecule must pass to go from A to B or vice versa. c)A reasonable path for the transition of a pyranose (such as glucose) from boat (1) to chair (3) conformation. The highest energy state—the transition state—will look something like (2). Effect of increasing temperature or lowering DGo+ on the rates of reactions: The rates of reactions are proportional to the number of molecules with sufficient energy to overcome the activation barrier DGo+. (a) At higher temperature more molecules have this energy. (b) Lowering the value of also increases the number of molecules with sufficient energy to attain the transition state.  The rate enhancement for an enzyme-catalyzed reaction is the ratio of the rate constants for the catalyzed (kcat) and the noncatalyzed (knon) reactions. The rate enhancement indicates how much faster the reaction occurs in the presence of the enzyme. Enzymatic rate enhancements: Logarithmic scale of kcat and knon values for some representative reactions at 25°C. The length of each vertical bar represents the rate enhancement achieved by the enzyme. ADC arginine decarboxylase; ODC orotidine 5’-phosphate decarboxylase; STN staphylococcal nuclease; GLU = sweet potato a-amylase; FUM fumarase; MAN mandelate racemase; PEP carboxypeptidase B; CDA E. coli cytidine deaminase; KSI ketosteroid isomerase; CMU chorismate mutase; CAN carbonic anhydrase.  The presence of a catalyst increases forward and reverse rates for a reaction, but does not affect the equilibrium composition of reactants and products. Effect of a catalyst on activation energy: The catalyst lowers the standard free energy of activation, DGo+, and thereby accelerates the rate because more of the reactant molecules have the energy needed to reach this lowered transition state. The rate enhancement is related to DDGo+. Note that the values of DGoA  B for both the catalyzed and noncatalyzed reactions are the same; thus, the reaction equilibrium is not perturbed by the presence of the catalyst.  How Enzymes Act as Catalysts: Principles and Examples An enzyme active site is complementary in shape, charge, and polarity to the transition state for the reaction, and to a lesser extent, its substrate. Enzyme-substrate complementarity is the basis for the specificity of enzyme-catalyzed reactions. Their catalytic activity depends on the integrity of their native protein conformation. If an enzyme is denatured or dissociated into its subunits, catalytic activity is usually lost. If an enzyme is broken down into its component amino acids, its catalytic activity is always destroyed. Thus the primary, secondary, tertiary, and quaternary structures of protein enzymes are essential to their catalytic activity Entropic and enthalpic factors in catalysis: In this example, two reactants are bound to sites on the catalyst which ensures their correct mutual orientation and proximity, and binds them most strongly when they are in the transition state conformation. Importance of intermediate states: An enzyme may alter the reaction pathway to one that includes one or more intermediate states that resemble the transition state but have a lower free energy. In the case of a single intermediate, the activation energies for formation of the intermediate state and for conversion of the intermediate to product are lower than the activation energy for the uncatalyzed reaction. Two models for enzyme–substrate interaction: In this example, the enzyme catalyzes a cleavage reaction. a) The lock-and-key model. In this early model, the active site of the enzyme fits the substrate as a lock does a key. b) The induced fit model. In this elaboration of the lock-and-key model, both enzyme and substrate are distorted on binding. The substrate is forced into a conformation approximating the transition state; the enzyme keeps the substrate under strain. Thus, an enzyme: (1) Binds the substrate or substrates (2) Lowers the energy of the transition state (3) Directly promotes the catalytic event  For an enzyme (E) that catalyzes the conversion of a single substrate (S) into a single product (P), the expression for the reaction includes three steps: ES is the enzyme–substrate complex, and EP is the enzyme bound to the product. For many enzyme-catalyzed reactions the first step, binding of substrate, is reversible (i.e., k1 and k-1 >> k2). The second step, conversion of ES to EP, lies far to the right (i.e., k2 >> k-2). The third step, release of product, is rapid compared to the catalytic step (i.e., k3 >> k2). The induced conformational change in hexokinase: The binding of glucose to hexokinase induces a significant conformational change in the enzyme. The enzyme is a single polypeptide chain, with two major domains. Notice how the obvious cleft between the domains (panel a) closes around the glucose molecule. Reaction coordinate diagram for a simple enzyme catalyzed reaction: Enthalpic stabilization of the transition state in an enzyme-catalyzed reaction: This panel shows the transition state and tetrahedral intermediate for an enzyme-catalyzed ester cleavage.  The transition state might be stabilized by electrostatic interactions with active site amino acids and/or metal ions, or it might be stabilized by general acids or bases. The direction of proton transfer from the proton donor to the proton acceptor is indicated by the placement of the wide end of the dashed bond near the proton acceptor. The GAC is a proton donor and the GBC is a proton acceptor.  The active site cleft of lysozyme: Top: The solvent-accessible surface of hen lysozyme is shown in blue. The trisaccharide NAM-NAG-NAM is shown in stick representation bound to the active site. Bottom: A schematic drawing of (NAG-NAM)3 bound to the A–F subsites in lysozyme. The substrate of lysozyme is peptidoglycan, a carbohydrate found in many bacterial cell walls. Lysozyme cleaves the (β1 → 4) glycosidic C—O bond between the two types of sugar residues in the molecule, acetylmuramic acid (Mur2Ac) and N-acetylglucosamine (GlcNAc) The mechanism of action of lysozyme: The Phillips mechanism is illustrated by the black reaction arrows along the left side of the diagram. In the first step, E35 acts as a general acid to promote cleavage of the glycosidic bond and concomitant formation of the oxocarbenium ion (which is stabilized electrostatically by D52). In the second step, E35 acts as a general base, deprotonating a water molecule, which then attacks C1 of the substrate. The mechanism of action of lysozyme: The pathway that includes the covalent intermediate reported by Steve Withers follows the green reaction arrows along the right side of the diagram. In this case, the second step involves covalent bond formation between C1 of the substrate and D52. Attack of the water displaces D52 in the subsequent step. The effect of pH on the activity of lysozyme: E35 must be protonated to act as a general acid catalyst in the first step of the mechanism; thus, at pH values below 6.2 the ratio of [COOH]/[COO- ] is greatest, favoring catalysis. D52 must be deprotonated to interact with the oxocarbenium ion; thus, at pH values above 3.7 the ratio of [COO-]/[COOH] is greatest, favoring catalysis. These two boundary requirements give rise to the observed pH optimum (~5) where both protonated E35 and deprotonated D52 are abundant. Evidence for the covalent intermediate in the mechanism of lysozyme: The covalent adduct between the synthetic substrate NAG-2FGlcF and D52 in the active site of the E35Q mutant of lysozyme is shown. Catalysis of peptide bond hydrolysis by chymotrypsin: Catalysis of peptide bond hydrolysis by chymotrypsin: The structure of chymotrypsin and the serine protease catalytic triad: The backbone of bovine chymotrypsin detemined by X-ray crystallopgraphy. Here, H57 is protonated, S195 and D102 are deprotonated.  The catalysis of peptide-bond cleavage by serine proteases involves stabilization of transition states and tetrahedral intermediate states. Dynamic conformational changes in the catalytic cycle of dihydrofolate reductase: