Biochem 4.3 Enzyme Catalysis PDF

Catalytic Mechanisms of EnzymesIntroductionAs discussed in Lesson 4.1, enzymes are catalysts that affect reaction rate and kinetics without altering thermodynamics and equilibrium. All catalysts share these properties. However, enzymes exhibit many additional properties that differentiate them from...

Catalytic Mechanisms of EnzymesIntroductionAs discussed in Lesson 4.1, enzymes are catalysts that affect reaction rate and kinetics without altering thermodynamics and equilibrium. All catalysts share these properties. However, enzymes exhibit many additional properties that differentiate them from chemical catalysts. These properties largely arise because enzymes are proteins and have the properties of proteins discussed in Chapters 2 and 3. This lesson elaborates on some unique properties of enzymes that arise from their biological (rather than chemical) nature and delves deeper into the catalytic mechanisms of enzymes.4.3.01 Enzyme-Substrate InteractionsAs discussed in Lesson 4.1, enzymes catalyze reactions by lowering their activation energy to increase the reaction rate.ReactantEnzymeProductEnzymes must physically interact with and bind to reactants before catalysis can occur. In other words, the reactant is a ligand that the enzyme acts upon by catalyzing its chemical change. Because the reactants of enzyme-catalyzed reactions are ligands that are changed through a chemical reaction, enzyme reactant ligands are given the special name of substrate (Figure 4.39).Figure 4.39 Ligands can bind to and unbind from proteins without being chemically changed. Substrates are ligands that bind to enzymes and are subsequently chemically changed.Ligand binding to proteins is stabilized by a variety of that depend on the functional groups of the ligand and the protein residues or backbone groups involved. These intermolecular interactions and the formation of noncovalent bonds result in a release of heat and a decrease in. and can also stabilize protein-ligand interactions through an Chapter 4: Enzyme Activity168increase in entropy of the surrounding water in the system. In this way, the of a system decreases when a protein binds its ligand until equilibrium is reached (∆Gbinding).Recall that enzymes catalyze reactions by decreasing the activation energy (Ea). Because enzymes do not affect equilibrium or thermodynamics, the free energy of the unreacted substrate is unchanged; therefore, the decrease in Ea must come from a decrease in the free energy of the transition state (‡). Stabilizing protein-ligand interactions lower the free energy of the enzyme--transition state complex compared to uncomplexed transition state, which leads to an increased reaction rate (Figure 4.40).Figure 4.40 Free energy diagrams of an uncatalyzed reaction (A) and an enzyme-catalyzed reaction (B). The activation energy Ea is greatly decreased by binding to the enzyme (∆Gbinding).Note that Figure 4.40B shows the enzyme in three bound states: one bound to the substrate, one bound to the product, and one bound to the transition state ‡. The enzyme-substrate and enzyme-product complexes have similar or higher free energies compared to the substrate and product alone. This relationship is important to ensure that substrates and products release from the enzyme (rather than staying bound).In contrast, the enzyme--transition state complex has a significantly lower free energy than the unbound transition state. This shows that the transition state is the species for which the binding interaction energies are strongest (ie, the shift from unbound transition state to bound transition state has the most negative ∆Gbinding of the various bound states shown). This implies that the optimal ligand for an enzyme is neither its substrate nor its product, but rather the transition state (Figure 4.41). Chapter 4: Enzyme Activity169Figure 4.41 Under physiological conditions, the binding energy ∆Gbinding is most negative (ie, most favorable) when the enzyme is bound to the transition state.Concept Check 4.9A medicinal chemist is designing a drug against Enzyme X. She plans to design a competitive inhibitor of the enzyme, which means the drug will bind the enzyme in place of the substrate, but it will not react. If the scientist wants to design a drug with the highest binding strength (ie, lowest Kd) possible, should she design the drug to most closely resemble the substrate, the product, or the transition state of the enzyme-catalyzed reaction? Chapter 4: Enzyme Activity170Concept Check 4.10After designing a candidate drug, structural analysis shows that a negatively charged functional group on the drug interacts strongly with a positively charged residue in the enzyme\'s substrate-binding site. Given this, which of the following mutant enzyme variants is likely to have the weakest interaction with the drug candidate? Assume all mutations are in the substrate-binding site.A.V56IB.K44LC.D24ED.R39K4.3.02 Lock-and-key versus Induced-Fit TheoriesConcept 4.3.01 describes how an enzyme\'s optimal ligand is the transition state of the enzyme-catalyzed reaction, yet to catalyze a reaction, an enzyme must also be able to recognize and bind to its substrates. How does an enzyme recognize the substrates it binds to, and what flexibility does an enzyme have to recognize related molecules? Several models have been proposed to address these questions.The lock-and-key model posits that even when not bound to its substrate, the enzyme displays a that perfectly complements (ie, matches) its specific substrate. Only the substrate of interest can perfectly fit into the substrate-binding pocket of the enzyme, like a key fitting into a lock, and no is needed to accommodate the substrate (Figure 4.42).Figure 4.42 The lock-and-key model of an enzyme binding its substrate. Chapter 4: Enzyme Activity171In contrast, the induced-fit model proposes that the resting enzyme does not perfectly complement the substrate. Instead, the substrate itself causes the enzyme to adopt a structure to accommodate it (Figure 4.43). In other words, the substrate induces a conformational change in the enzyme to fit around the substrate (see Concept 2.3.03 for more on conformational changes).Figure 4.43 The induced-fit model of an enzyme binding its substrate.A third model, which is related to the induced-fit model, is conformational selection. In this model, the unbound enzyme explores a range of conformations near the low point of its (see Concept 2.3.02). Although the predominant conformation is the low-binding-affinity conformation (ie, a conformation that does not fit the substrate), at any given time a of enzyme is in the high-binding-affinity conformation (ie, the conformation that does fit substrate).Rather than the substrate inducing a conformational change, the conformational selection model proposes that the substrate binds to one of the few proteins that has temporarily adopted this high-affinity conformation. Upon binding, the high-affinity conformation is stabilized. As more substrate is added, a higher percentage of the total enzyme population becomes stabilized in this high-affinity conformation (Figure 4.44). Chapter 4: Enzyme Activity172Figure 4.44 The conformational selection model of an enzyme binding its substrate.Given the three different models of molecular recognition presented, which model is relevant to enzyme-substrate binding? Although some enzymes may exhibit features of one model over the others, all three models contribute useful insight into understanding enzyme-substrate recognition (and protein-ligand recognition in general), and most enzymes can be explained by some mix of all three models.Concept Check 4.11For each of the three models of molecular recognition (lock-and-key model, induced-fit model, conformational selection), place the following steps in sequential order, starting from unbound enzyme and ending in high-affinity substrate binding. Not all models will require all steps.A.Enzyme is in the unbound ground state.B.Substrate binds the enzyme with high affinity.C.Substrate binds the enzyme with low affinity.D.Enzyme undergoes a conformational change.4.3.03 Implications of the Molecular Recognition ModelsThe lock-and-key model was first proposed as a way of explaining the specificity of an enzyme for its substrate. Specificity refers to the high selectivity with which an enzyme acts on its own substrate, but not on other substrates with related structures. For example, many proteases (enzymes that hydrolyze peptide bonds) act on only specific amino acid sequences. Rather than acting on all peptide bonds, proteases accommodate only certain substrates that fit their binding pocket (Figure 4.45). Chapter 4: Enzyme Activity173Figure 4.45 Enzymes can be highly specific for their substrates and may fail to act on molecular structures that are very similar to the natural substrate.Similarly, most amino acids and peptides found in nature display highly specific , as discussed in Unit 1. Most amino acids are chiral, with most physiologically relevant amino acids being the ʟ-enantiomer. Consequently, most proteins (including enzymes) are chiral as well. This leads to one of the important distinctions between chemical catalysts and biological catalysts such as enzymes. Whereas chemical catalysts are mostly not stereoselective, enzymes and other biological catalysts usually are highly stereoselective. An enzyme that acts on one isomer of a molecule often does not act on other isomers of the same molecule (Figure 4.46). Chapter 4: Enzyme Activity174Figure 4.46 Most enzymes are stereoselective so that only the correct stereoisomer (the \"key\") fits into the substrate-binding pocket (the \"lock\").Although the lock-and-key model can explain the high specificity of some enzymes, it does not adequately explain why other enzymes are less specific and can recognize a larger variety of substrates. Furthermore, it does not adequately explain an important feature discussed in Concept 4.3.01---that the optimal ligand for an enzyme is not the substrate, but rather the transition state of the enzyme-catalyzed reaction.Both the induced-fit model and the conformational selection model allow for conformational changes in the enzyme, which may permit some enzymes to act on multiple related substrates because they allow for conformational adjustments of the substrate-binding site. Furthermore, these models help explain how a short-lived transition state can be the optimal ligand of the enzyme. Just as the substrate induces a conformational change in the enzyme, the enzyme also induces a conformational change in the substrate toward its transition state (Figure 4.47). Chapter 4: Enzyme Activity175Figure 4.47 Both the induced-fit model (left) and the conformational selection model (right) help explain how enzymes recognize a transition state.4.3.04 Enzyme Active SiteThe previous concepts in this lesson discuss how enzymes recognize and interact with their substrate through the formation of noncovalent bonds. The decrease in enthalpy H associated with the formation of these bonds results in the stabilization of the reaction\'s transition state, which results in the enzyme\'s ability to catalyze the reaction. In other words, the binding energy contributes to the activity of the enzyme by helping decrease the activation energy Ea. For this reason, the substrate-binding site of an enzyme contributes a crucial component of an enzyme\'s active site (Figure 4.48). Chapter 4: Enzyme Activity176Figure 4.48 The active site of an enzyme is the portion of the enzyme where substrates bind and where catalysis of the reaction occurs.In addition to binding, some enzymes can employ additional mechanisms to catalyze chemical reactions. These mechanisms, which are covered in more detail in Concept 4.3.06, often require changes in the chemical reactivity of specific enzyme residues. For example, Concept 1.3.06 discusses how the of an can be , yet the pKa values for free amino acids suggest that the side chains of most nucleophilic amino acids are protonated at physiological pH.Concept 2.1.04 introduces the idea that the pKa of an amino acid residue can change due to interactions with nearby functional groups. The tertiary structure of the enzyme creates a microenvironment that often brings specific functional groups together, allowing the nucleophilic amino acid to lose a proton at physiological pH values.As an example, the catalytic triad in many serine proteases is a set of three amino acids in the enzyme active site that work together to allow a serine side chain to act as a nucleophile. Although serine\'s high pKa (around 13) makes the residue unlikely to lose a proton in aqueous solution, the unique microenvironment of the active site forces a basic histidine residue into close proximity with the serine side chain. This allows serine to act as an acid by giving up its proton to the basic histidine, thereby enhancing serine\'s nucleophilicity. The protonated histidine, in turn, is stabilized by the third member of the catalytic triad: an acidic residue such as aspartate (Figure 4.49). Chapter 4: Enzyme Activity177Figure 4.49 The catalytic triad is an example of residues contributing to a unique microenvironment within an enzyme active site.Concept Check 4.12Amino groups (R--NH3+) can often act as nucleophiles in biochemical systems. For example, the α-amino group of an amino acid acts as a nucleophile during peptide bond formation, and the side-chain ε-amino group of lysine acts as a nucleophile during the isopeptide bond formation reactions of ubiquitination or histone acetylation.A novel enzyme has been found to catalyze isopeptide bond formation, and the side chain amino group of the substrate lysine is found to interact strongly with specific active site residues. The lysine side chain amino group is most likely to interact with the side chain of which of the following amino acids in the enzyme active site to enhance its nucleophilicity?A.LeucineB.HistidineC.GlycineD.Methionine Chapter 4: Enzyme Activity1784.3.05 Enzymes with Multiple SubstratesImportantly, many enzymes catalyze reactions with multiple substrates. Despite this, many enzyme-catalyzed reactions occur at a single active site. In these instances, the active site may have multiple substrate-binding subsites that hold the substrates and a singular catalytic subsite where covalent bonds are formed and broken. Depending on the specific case, an enzyme may hold multiple substrates at the same time, or it may react with the substrates one by one.An enzyme that holds two substrates at the same time is said to form a ternary complex because it is a complex of three distinct molecules: one enzyme and the two substrates together. Some enzymes that form ternary complexes may bind their substrates in an ordered manner (ie, substrate A must bind before substrate B). Other enzymes that form ternary complexes bind their substrates in a random order (ie, the enzyme can accept either substrate A or substrate B first (see Figure 4.50).Figure 4.50 A ternary complex is a three-membered complex.In contrast, other enzymes bind and react with one substrate in one step---forming and releasing one product and yielding a modified enzyme as a reaction intermediate---before the modified enzyme binds and reacts with the second substrate. After the second step, the second product is released. As discussed in Concept 4.1.05, enzymes must be unaltered at the end of the reaction, so this second step must also restore the enzyme to its original state. Enzymes that act in this manner are said to follow a double displacement or ping-pong mechanism (Figure 4.51). Chapter 4: Enzyme Activity179Figure 4.51 A double displacement, also known as ping-pong, enzyme reaction mechanism.Enzymes may also have multiple active sites that each catalyze separate reactions. These sites are usually found on separate domains or subunits and act independently of each other. For example, the bifunctional enzyme phosphofructokinase-2/fructose-2,6-bisphosphatase (see Chapter 11) has separate active sites on different domains that catalyze separate kinase and phosphatase reactions. Similarly, fatty acid synthase (see Concept 13.1.04) also contains several active sites on different domains to catalyze the various reactions involved in fatty acid synthesis.Cooperative enzymes also display multiple active sites that each catalyze several instances of the same reaction (see Concept 5.3.05). However, the term \"multisubstrate reaction\" (eg, ternary complex or double displacement) refers to a reaction involving multiple substrates at a single active site, not to multiple active sites acting separately. Chapter 4: Enzyme Activity180Concept Check 4.13Transaminases are enzymes that catalyze reactions such as the one shown in the following image:A scientist mixes aspartate with the transaminase enzyme. After a brief incubation period, the scientist detects a very small amount of oxaloacetate, despite not having added any α-ketoglutarate. Based on this information, does the transaminase enzyme form a ternary complex, or does it proceed by a double displacement (ping-pong) mechanism? If instead the scientist tested a different two-substrate enzyme but was not able to detect any product formation after addition of a single substrate, what could be concluded about the mechanism in this alternate scenario?4.3.06 Modes of CatalysisEffects of Binding, Conformational Changes, and Other Physical EffectsAs mentioned in Concept 4.3.01, binding energies between the enzyme and the reaction transition state ‡ lead to a lower free energy G of the enzyme--transition state complex compared to free transition state. This decrease in free energy (and therefore the decrease in activation energy Ea) is a result of negative enthalpy changes (−∆H) due to formation of noncovalent, stabilizing bonds between the enzyme and the transition state, as well as increases in entropy (∆S) due to.Although the formation of a large enzyme--transition state complex may at first glance seem like an increase in order, which should result in a decrease in entropy, substrate binding forces water to leave the enzyme active site, causing the water molecules to become more disordered, which leads to an increase in entropy for the whole system (Figure 4.52). This desolvation effect is similar to the hydrophobic effect discussed in Lesson 2.3. Chapter 4: Enzyme Activity181Figure 4.52 Entropy and enthalpy changes contribute to a decreased free energy of the enzyme--transition state complex (decreased Ea).In addition to the binding energy itself, stabilization of the transition state in the enzyme also results in increased strain of the substrate bonds relative to unreacted substrate. The transition state normally exists only temporarily because this strain gives it a high free energy. Although the enzyme stabilizes the free energy, the strain still primes the substrate for conversion into product (Figure 4.53). Chapter 4: Enzyme Activity182Figure 4.53 The strain in the transition state\'s bonds primes the substrate for conversion into product.Enzymes also speed up reactions by physically positioning functional groups in ways that more easily result in productive collisions. proposes that chemical reactions only occur if substances collide in the correct orientation with sufficient energy to overcome Ea.By binding substrates at a single active site, enzymes effectively increase the local concentration of reactants, increasing frequency of collisions. The specific shape of the active site also forces substrates to bind in a specific way, ensuring that the collisions that do occur happen in the correct orientation (Figure 4.54). Chapter 4: Enzyme Activity183Figure 4.54 Enzymes help catalyze reactions by holding the reacting functional groups in close proximity and in the correct orientation.Covalent CatalysisConcept 4.3.04 introduces the catalytic triad of a serine protease as an example of a mechanism in which an enzyme\'s active site contributes to a microenvironment that changes functional group protonation state and reactivity. In the catalytic triad, a serine residue of the enzyme active site is deprotonated, allowing it to act as a nucleophile and attack the substrate, forming a covalent bond. Because the enzyme forms a temporary covalent bond with a substrate, this is referred to as covalent catalysis.Covalent catalysis can speed up reactions by converting stable functional groups into less stable functional groups. Consider the hydrolysis of a peptide bond by a serine protease. The uncatalyzed hydrolysis reaction involves a water molecule nucleophilically attacking the peptide bond. However, peptide bonds are a subset of amide bonds, which are not very reactive due to. Therefore, peptide bonds are not easily attacked by a weak nucleophile such as water, even in an enzyme active site.In contrast, a deprotonated alcohol like serine is a strong nucleophile that can break the amide bond. Doing so replaces the amide with an ester, which can be by water (Figure 4.55). Chapter 4: Enzyme Activity184Figure 4.55 Covalent catalysis occurs when the enzyme makes a temporary covalent bond with its substrate.Note that this covalent catalysis mechanism increases the number of steps needed to complete the reaction. Because free energy is a state function (as described in Lesson 4.1) this does not affect the overall free energy change ∆G of the reaction.However, it does change the reaction from one with a single large Ea representing one transition state to a reaction with two smaller Ea values representing two transition states. This example also demonstrates that enzymes do not necessarily stabilize the transition state of the uncatalyzed reaction but rather may produce distinct transition state(s) (Figure 4.56). Chapter 4: Enzyme Activity185Figure 4.56 Enzyme-catalyzed reactions can proceed through a different reaction pathway than uncatalyzed reactions, which may result in different transition states.Acid-Base CatalysisThe catalytic triad also demonstrates acid-base catalysis, wherein enzymes catalyze reactions by changing the protonation state, and therefore the reactivity, of various functional groups. In the case of the catalytic triad, the protonation state of the enzyme itself---specifically its active site serine residue---is altered by the active site microenvironment; however, many enzymes can also create an active site microenvironment that affects the substrate\'s protonation state.If the enzyme itself serves as the Brønsted-Lowry acid (proton donor) or base (proton acceptor), this is known as general acid-base catalysis. In contrast, when hydronium or hydroxide ions from solution are used as the acid or base, this is known as specific acid-base catalysis. Because histidine\'s pKa of 6 is very close to the physiological pH of 7.4, histidine often plays an important role in general acid-base catalysis, although other residues may also contribute depending on the active site microenvironment.Effects of Cofactors and CoenzymesConcept 2.4.03 introduced and coenzymes as important non--amino acid components of proteins. Many enzymes also utilize cofactors or coenzymes in their catalytic mechanism. Metal ions are common inorganic cofactors that assist in catalysis by electrostatically stabilizing the substrate and positioning it in the correct orientation, by serving as a or by acting as a.Coenzymes are organic cofactors and include molecules such as nicotinamide adenine dinucleotide (NAD+ and NADH), flavin adenine dinucleotide (FAD and FADH2), coenzyme A (CoA-SH), and coenzyme Q (ubiquinone and ubiquinol). The large variety of coenzymes indicates the large number of roles coenzymes can play. Figure 4.57 depicts the use of several coenzymes in the pyruvate dehydrogenase complex. Chapter 4: Enzyme Activity186For some enzymes, the coenzyme is unchanged by the end of the reaction, and the coenzyme truly serves a catalytic role. Other enzymes modify a coenzyme such that the coenzyme does not revert to its original state by the end of the reaction. For this reason, such coenzymes are more accurately described as co-substrates; nevertheless, the name coenzyme persists, likely because the molecules are eventually regenerated after several metabolic enzyme-catalyzed reactions in other pathways.Figure 4.57 The pyruvate dehydrogenase complex is an example of an enzyme complex that uses multiple coenzymes.Figure 4.57 shows the pyruvate dehydrogenase enzyme complex (PDH), which catalyzes an intermediate step between glycolysis and the citric acid cycle (see Unit 4), as an example of this coenzyme usage. PDH uses the coenzymes thiamine pyrophosphate (TPP), lipoic acid (Lip), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD+). TPP, Lip, and FAD are restored to their original state by the end of the reaction. However, NAD+ remains converted to NADH at the end of the reaction.