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Chap. 6B Enzymes. Introduction to Enzymes How Enzymes Work Enzyme Kinetics as an Approach to Understanding Mechanism Examples of Enzymatic Reactions Regulatory Enzymes. Fig. 6-22. The chymotrypsin enzyme-substrate complex. Intro. to Enzyme Mechanisms.
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Chap. 6B Enzymes • Introduction to Enzymes • How Enzymes Work • Enzyme Kinetics as an Approach to Understanding Mechanism • Examples of Enzymatic Reactions • Regulatory Enzymes Fig. 6-22. The chymotrypsin enzyme-substrate complex.
Intro. to Enzyme Mechanisms An understanding of the complete mechanism of action of a purified enzyme requires identification of all substrates, cofactors, products, and regulators. Moreover, it requires a knowledge of 1) the temporal sequence in which enzyme-bound reaction intermediates form, 2) the structure of each intermediate and each transition state, 3) the rates of interconversion between intermediates, 4) the structural relationship of the enzyme to each intermediate, and 5) the energy contributed by all reacting and interacting groups to intermediate complexes and transition states. As yet, there is probably no enzyme for which all of these features are completely known. However, many of these mechanistic aspects have been established for enzymes such as chymotrypsin, which will be the focus of our discussion of enzyme mechanisms.
The Structure of Chymotrypsin (CT) Bovine pancreatic chymotrypsin (Mr 25,191) is a protease, or enzyme that catalyzes the hydrolytic cleavage of peptide bonds in target proteins. CT is specific for peptide bonds adjacent to aromatic amino acid residues (Trp, Phe, & Tyr). Cleavage occurs on the C-terminal side of these residues. The three-dimensional structure of CT is shown in Fig. 6-19, with functional groups in the active site emphasized. The protein consists of three polypeptide chains linked by disulfide bonds. The three chains are derived by proteolytic processing of a slightly longer preprotein known as chymotrypsinogen, as explained in Fig. 6-38, below). The key catalytic residues of the active site (His57, Asp102, Gly193, and Ser195 of the primary structure) are brought together in three-dimensional space in the tertiary structure. The hydrophobic pocket in which the aromatic amino acid side-chain of the substrate is bound is highlighted in yellow.
Intro. to the CT Reaction Mechanism (I) The reaction catalyzed by CT illustrates the principle of transition state stabilization and also provides a classic example of general acid-base catalysis and covalent catalysis. CT enhances the rate of peptide bond hydrolysis by a factor of at least 109. It does not catalyze a direct attack of water on the peptide bond. Instead, a transient covalent acyl-enzyme intermediate is formed. The reaction has two distinct phases. In the acylation phase, the peptide bond is cleaved and an ester linkage is formed between the peptide carbonyl carbon and the enzyme. In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated enzyme is regenerated. As covered below, the nucleophile in the acylation phase is the oxygen atom in the side-chain of Ser195. Proteases with serine residues that play this role in reaction mechanisms are called serine proteases. The pKa of a serine hydroxyl group is generally too high for the unprotonated form to be present in significant concentrations at physiological pH. However, in CT, Ser195 is linked to His57 and Asp102 in a hydrogen-bonding network referred to as the catalytic triad. The association of His57 and Asp102 raises the pKa of the His residue to > 12, allowing the His to act as an enhanced general base that can remove the proton from Ser195 creating an extremely reactive nucleophile. At later reaction stages, His57 acts as a proton donor, protonating the amino group in the displaced portion of the substrate (the leaving group).
Intro. to the CT Reaction Mechanism (II) As the Ser195 oxygen attacks the carbonyl group of the substrate, a very short-lived tetrahedral intermediate is formed in which the carbonyl oxygen acquires a negative charge (Fig. 6-22, Step 2). This charge, forming within a pocket on the enzyme called the oxyanion hole, is stabilized by hydrogen bonds to the CT backbone. One of these hydrogen bonds is contributed by Gly193 and is present only in this intermediate and in the transition state for its formation and breakdown. This interaction reduces the activation energy required to reach these states. This is an example of the use of binding energy in catalysis.
Evidence for an Acyl-enzyme Intermediate The first evidence for a covalent acyl-enzyme intermediate came from a classic application of pre-steady state kinetics. In addition to its action on polypeptides, CT also catalyzes the hydrolysis of small esters and amides. These reactions are much slower than peptide hydrolysis because less binding energy is available with smaller substrates, and they are therefore easier to study. CT hydrolysis of the ester p-nitrophenylacetate, as measured by the release of p-nitrophenol, proceeds with a rapid burst before leveling off to a slower rate (Fig. 6-20). By extrapolating back to zero time, the burst phase corresponds to just under one molecule of p-nitrophenol released for every enzyme molecule present. This is consistent with the rapid acylation of all enzyme molecules, with the rate for subsequent turnover of the enzyme limited by a slower deacylation step.
pH Dependence of the CT Reaction The rate of CT-mediated cleavage produces a bell-shaped pH-rate profile with an optimum at pH 8.0 (Fig. 6-21). The rate (v) being plotted is that at low substrate concentrations and thus reflects the term kcat/Km. The plot can be broken down to its components using kinetic methods to determine the terms kcat and Km separately at each pH (not discussed). When this is done (Parts b & c), it becomes clear that the transition just above pH 7 is due to changes in kcat, whereas the transition above pH 8.5 is due to changes in 1/Km. Kinetic and structural studies have shown that the transitions illustrated in Parts (b) and (c) reflect the ionization states of the His57 side-chain (when substrate is not bound) and the -amino group of Ile16 at the amino terminus of the B chain, respectively. For optimal activity, His57 must be unprotonated so it can remove the hydrogen atom from Ser195 at the start of the CT reaction. Ile16 must be protonated for binding of the substrate, hence the effect on 1/Km.
CT Mechanism: Acylation Phase (Step 1) The next few slides cover the acylation and deacylation phases of the CT reaction mechanism. These slides are derived from Fig. 6-22.
Review of Nucleophiles and Electrophiles in Biochemistry In many reactions of biochemistry, an electron-rich atom (a nucleophile) reacts with an electron-deficient atom (an electrophile). Some common nucleophiles and electrophiles in biochemistry are shown in the diagram below.
Intro. to Regulatory Enzymes Regulatory enzymes exhibit increased or decreased catalytic activity in response to certain signals. Regulatory enzymes play important roles in governing the rate of flux of compounds through metabolic pathways in cells. The activities of regulatory enzymes are modulated in a number of ways. Allosteric enzymes are regulated by the reversible, noncovalent binding of regulatory molecules called allosteric modulators or effectors. Another type of regulation occurs via reversible covalent modification of enzyme side-chains. Both types of regulatory enzymes tend to be multisubunit proteins. In many cases the regulatory sites and the active site are on separate subunits. Two other types of regulation also are common. These are regulation achieved by the binding of a separate regulatory protein to the enzyme of interest, and proteolytic cleavage of the target enzyme, removing peptide segments that inhibit its activity. Unlike effector-mediated regulation, regulation by proteolytic cleavage is irreversible. Note that more than one type of regulation (e.g., allosteric and covalent) can occur for a single regulatory enzyme.
Conformational Changes in Allosteric Regulatory Enzymes The modulators of an allosteric enzyme may be inhibitory or stimulatory. If the modulator and substrate are the same the regulation is called homotropic. If the modulator and substrate are different molecules the regulation is called heterotropic. Allosteric enzymes generally have one or more regulatory, or allosteric, sites for binding the modulator. Just as an enzyme’s active site is specific for its substrate, each regulatory site is specific for its modulator. In many allosteric enzymes, the substrate-binding and modulator-binding sites(s) are on different subunits. These are called the catalytic (C) and regulatory (R) subunits. In Fig. 6-32, binding of the positive (stimulatory) modulator (M) to its specific site on the regulatory subunit is communicated to the catalytic subunit through a conformational change. This change renders the catalytic subunit active and capable of binding the substrate (S) with higher affinity. On dissociation of the modulator from the regulatory subunit, the enzyme reverts to its inactive or less active form
Regulation of Aspartate Transcarbamoylase (I) A classic example of a regulatory enzyme is aspartate transcarbamoylase (ATCase). This enzyme catalyzes a regulatory step early in the biosynthesis of pyrimidine nucleotides. In this reaction, carbamoyl phosphate and aspartate combine to form N-carbamoyl aspartate.
Regulation of Aspartate Transcarbamoylase (II) ATCase has 12 polypeptide chains organized into 6 catalytic (organized as 2 trimeric complexes) and 6 regulatory (organized as 3 dimeric complexes) subunits (Fig. 6-33). The regulatory subunits have binding sites for ATP and CTP, which function as positive and negative regulators, respectively. CTP is one of the end products of the pyrimidine nucleotide biosynthetic pathway, and negative regulation by CTP serves to limit ATCase activity under conditions when CTP is abundant. High concentrations of ATP indicate that cellular metabolism is robust, the cell is growing, and additional pyrimidine nucleotides may be needed to support RNA transcription and DNA replication. As shown in the diagram, the binding of the negative modulator CTP to the regulatory subunits produces large changes in enzyme conformation, and converts the complex to the less active T state.
Kinetic Properties of Regulatory Enzymes (I) Allosteric enzymes show relationships between V0 and [S] that differ from MM kinetics. They do exhibit saturation with the substrate when [S] is sufficiently high, but for allosteric enzymes, plots of V0 versus [S] usually produce a sigmoid saturation curve, rather than the hyperbolic curve typical of nonregulatory enzymes. On the sigmoid saturation curve, a value of [S] at which the V0 is half-maximal can be identified, but it cannot be referred to as the Km because the enzyme does not follow the hyperbolic MM relationship. Instead the symbol [S]0.5 or K0.5 is often used to represent the substrate concentration giving half-maximal velocity for the reaction catalyzed by an allosteric enzyme. Sigmoid kinetic behavior generally reflects cooperative interactions between multiple protein subunits. Changes in the structure of one subunit are translated into structural changes in adjacent subunits due to changes in noncovalent interactions at the interfaces between subunits. Sigmoid kinetic behavior is explained by the concerted and sequential models for subunit interactions (Fig. 5-15).
Kinetic Properties of Regulatory Enzymes (II) Substrate-activity curves for representative allosteric enzymes that show complex responses to their modulators are shown in Fig. 6-34. In Panel (a), the sigmoid curve is for a homotropic enzyme, for which the substrate also serves as a positive modulator. This curve (black) resembles the oxygen-saturation curve for hemoglobin (Fig. 5-12). The sigmoidal curve is a hybrid curve in which the enzyme is present primarily in the relatively inactive T state at low substrate concentration, and primarily in the more active R state at high substrate concentration. The curves for pure T and R states are plotted separately in color. ATCase exhibits a kinetic pattern similar to this in response to the concentrations of its substrates, aspartate and carbamoyl phosphate.
Kinetic Properties of Regulatory Enzymes (III) In Panel (b) of Fig. 6-34, curves are plotted showing the effects of several different concentrations of a heterotropic positive modulator (+) and a heterotropic negative modulator (-) on an allosteric enzyme in which K0.5 is altered without a change in Vmax. The central curve shows the substrate-activity relationship in the absence of modulators. The curves shown are similar for ATCase in the presence of the negative modulator CTP and the positive modulator ATP.
Kinetic Properties of Regulatory Enzymes (IV) In Panel (c) of Fig. 6-34, curves are plotted showing the effects of modulators that change Vmax without significantly changing K0.5. This is a less common type of allosteric regulation, and it is not observed for ATCase.
Regulation by Reversible Covalent Modification For many enzymes, activity also is regulated by reversible covalent modification of one or more amino acid residues in the enzyme. Over 500 different types of covalent modifications have been found in proteins. Common modifying groups are shown in Fig. 6-35 and include phosphorylation, adenylylation, methylation, myristoylation, and ubiquitination. In the case of ubiquitination, the small protein ubiquitin is attached to a lysine residue in the target protein flagging it for proteolytic destruction. The modifying groups are usually attached to a regulated enzyme by a separate modifying enzyme (e.g., a methylase in the case of methylation). Introduction of the modifying group alters the local properties of the enzyme which typically undergoes a change in conformation and activity. Introduction of a hydrophobic myristoyl group triggers the association of the protein with a membrane. Phosphorylation is the most important type of covalent modification, and it is discussed further in subsequent slides.
Regulation by Reversible Phosphorylation: Muscle Glycogen Phosphorylase (I) It is estimated that one-third of all proteins in a eukaryotic cell are phosphorylated, and one, or often many phosphorylation events are part of virtually every regulatory process. Some proteins have only one phosphorylation site, whereas others have several, and a few have dozens of sites for phosphorylation. The attachment of phosphoryl groups to specific amino acid residues of a protein is catalyzed by enzymes called protein kinases. In these reactions, typically the -phosphoryl group from a nucleoside triphosphate (usually ATP) is transferred to a particular Ser, Thr, Tyr, or occasionally His residue in the target protein. Removal of the same phosphoryl group from the protein is performed by enzymes called protein phosphatases. The introduced phosphoryl group can cause major changes in the conformation of the modified enzyme by virtue of steric effects, or hydrogen bonding and ionic interactions to neighboring residues. This can lead to effects on substrate binding and catalysis.
Regulation by Reversible Phosphorylation: Muscle Glycogen Phosphorylase (II) An important example of enzyme regulation by phosphorylation is seen with glycogen phosphorylase of muscle and liver. This enzyme catalyzes the breakdown of glycogen stores via the reaction Glycogen(n) + Pi glycogen(n-1) + glucose 1-P In the liver, the released glucose 1-P is typically converted to free glucose and released to the bloodstream. In skeletal muscle, glucose 1-P is metabolized via the glycolytic pathway for energy production. Glycogen phosphorylase is a homodimeric enzyme that exists in two forms--the less active phosphorylase b form and the more active phosphorylase a species (Fig. 6-36). Activation by phosphorylation is catalyzed by the enzyme phosphorylase kinase which attaches phosphate groups to serine residues (Ser14) located near the N-termini of both subunits. The enzyme phosphoprotein phosphatase 1 (PP1) removes these two phosphates by hydrolysis, inactivating the enzyme. The phosphorylation of Ser14 causes major structural changes near the N-termini of the glycogen phosphorylase chains that produce a more active enzyme. Note that glycogen phosphorylase is also regulated by noncovalent allosteric modulation (Fig. 6-42, not covered).
Multiple Phosphorylations Allow Exquisite Regulatory Control The Ser, Thr, and Tyr residues that are typically phosphorylated in regulatory proteins occur within common structural motifs, called consensus sequences, that are recognized by specific protein kinases. For example the important kinase, protein kinase A, recognizes Ser/Thr residues in the consensus sequence -x-R-[RK]-x-[ST]-B-, where B is any hydrophobic amino acid (Table 6-10, not shown). Some proteins, such as glycogen synthase (Fig. 6-37), have consensus sequences recognized by several different protein kinases. In some cases, phosphorylation is hierarchical: a certain residue can only be phosphorylated if a neighboring residue has already been phosphorylated. Glycogen synthase, for example, is not a substrate for glycogen synthase kinase 3 until one site has been phosphorylated by casein kinase II. Some phosphorylations inhibit glycogen synthase more than others, and some combinations of phosphorylations have a cumulative effect on activity. The multiple regulatory phosphorylations provide the potential for finely tuned modulation of enzyme activity by controlling signals, such as hormones.
Regulation By Proteolytic Cleavage A number of proteolytic enzymes are synthesized in inactive forms called zymogens to control their catalytic activity until needed. When activity is needed they are then cleaved by an activating protease. Many proteolytic enzymes of the pancreas and stomach are regulated this way. For example, chymotrypsin and trypsin are initially synthesized as the zymogens chymotrypsinogen and trypsinogen (Fig. 6-38). Specific cleavages remove residues from these zymogens and expose the active sites. Because cleavage is irreversible, other mechanisms are needed to inactivate these enzymes. For example, proteases such as trypsin are inactivated by the tight binding of the inactivator protein, pancreatic trypsin inhibitor. Proteases are not the only proteins activated by proteolysis. Such other proteins are not called zymogens, but instead are called proproteins or proenzymes. For example, collagen is initially synthesized as the soluble precursor procollagen which is proteolytically processed to collagen chains.
