PAPER 5: MOLECULAR ENZYMOLOGY & PROTEIN ENGINEERING Module 17: Regulation and Feedback control

Introduction: The activity of proteins/ must be regulated so that they function at the proper time and place. The control of enzyme activity is essential in maintaining the steady state of all organisms. The functional expression of particular set of enzymes appears and disappears depending on the cellular need. Therefore, all the biochemical pathways, which can takes place in the cells does not operate in full swing at any given time. Rather, these pathways consisting of enzymatic reactions are regulated in very controlled manner to give optimal function and maintain the homeostasis. For example, glycogen synthesis and breakdown, both the processes occur in liver cells are tightly regulated and work exactly opposite to each other. Depending on the physiological requirement the systemic signals starts glycogen synthesis in liver cells however, when body runs out of energy source, it sends the signal to liver cells for the degradation of stored glycogen. Therefore, both these biochemical processes which acts against each other can takes place in same cells but not at the same time. The disruption of this highly ordered enzymatic control can disturb the homeostasis and can leads to severe pathological conditions.

Objectives:

In this chapter, we intend to learn the following aspects of Enzyme activity

 Mechanisms of Control of Enzymatic Activity o Substrate and product concentration o Protein turnover o By expressing various forms of same enzyme o Reversible Post translational Modification o Proteolytic control o Allosteric control  Feedback Inhibition  Kinetic Behavior of Allosteric Enzymes o Competetive and Non-competitive Inhibition  Mechanism of Allosteric Interactions

17.1 Mechanisms of Control of Enzymatic Activity

There are variety of mechanisms exist for regulating and controlling the enzyme activity as and when required. The regulation of enzymatic activity and its biochemical reactions starts with synthesis of enzyme (protein) itself. In several proteins/enzymes the activity is regulated by the conversion of enzymes from either “pro” form to “active” form or by posts translational modifications viz. phosphorylation, acetylation etc. On time scale, some of the enzyme activity regulation takes long time; like in cells, the induction of protein synthesis is a complex multistep process that typically requires hours to produce significant changes in overall enzyme level. However, changes in intrinsic catalytic efficiency effected by binding of dissociable ligands () or by covalent modification achieve regulation of enzymic activity within seconds. Changes in protein level serve long-term adaptive requirements, whereas changes in catalytic efficiency are best suited for rapid and transient alterations in metabolite flux. The biological activity of Enzymes/proteins is regulated in following ways:

17.1.1 Substrate and Product Concentration: The availability of substrate plays an important role in controlling the enzymatic activity. More concentration of substrate ensures more collision between the substrate and enzyme leading to E+S complex. The rate of reaction of most of the enzymes are responsive to changes in substrate concentration because the intracellular level of many substrates is in the range of the Km. Thus, an increase in substrate concentration prompts an increase in reaction rate, which tends to return the concentration of substrate toward normal. Usually, in any given biochemical pathway consists of many enzymatic reactions in a sequence, only few are controlled/ regulated. For example, the breakdown of glucose in to pyruvate () occurs in 10 steps, however the first reaction carried out by and few more are controlled. They are called rate limiting enzymes. The regulation of rate limiting enzymes occurs in order to conserve energy and metabolites. Sometimes, when the concentration of product is more, the enzymatic reaction gets inhibited and this phenomenon is called feedback inhibition. The first reaction of glycolysis, carried out by hexokinase is very good example of feedback inhibition. Hexokinase phosphorylates glucose to glucose-6-phosphate (G-6-P). If, G- 6-P is not utilized in further consecutive glycolytic reactions, its concentration will increase. Further, it will bind to hexokinase and stops the glucose phosphorylation reaction. This control of enzymatic activity by the end product of the reaction is referred to as negative feedback control.

Figure 17a: The diagram depicts, how the substrate and product concentration affects the equilibrium constants Ksyn. And Kdeg., which further controls the enzymatic activity.

17.1.2 Protein Turnover: The first determinant of enzyme activity is the level of enzyme/protein. Enzymes whose concentrations remain essentially constant over time are termed constitutive enzymes viz. metabolic enzyme, Glyceraldehyde-3-phosphate and structural protein b-Actin. However, the concentrations of many other enzymes depend upon the availability of inducers, substrates or structurally related compounds which initiates their synthesis, for example Escherichia coli grown on glucose will only catabolize lactose after addition of a β-galactoside, an inducer that initiates synthesis of a β-galactosidase and a galactoside permease. Inducible enzymes of humans include tryptophan pyrrolase, threonine dehydrase, tyrosine-α-ketoglutarate aminotransferase, enzymes of the urea cycle, HMG-CoA , and . On the other hand, an excess of enzyme product synthesis may suppress the enzyme synthesis. Both induction and repression involve cis elements, specific DNA sequences located upstream of regulated genes, and trans-acting regulatory proteins. The absolute quantity of an enzyme reflects the net balance between enzyme synthesis and enzyme degradation (shown in figure 17a). Protein turnover represents the net result of enzyme synthesis and degradation. Enzyme levels in mammalian tissues respond to a wide range of physiologic, hormonal, or dietary factors. For example, glucocorticoids increase the concentration of tyrosine aminotransferase by stimulating the protein synthesis. Regulation of liver can involve changes in protein synthesis and degradation. After a protein-rich meal, liver arginase levels rise and arginine synthesis decreases. Arginase levels also rise in starvation, but here arginase degradation decreases.

17.1.3 By expressing various form of same enzyme: During evolution, developed organisms evolved with various forms of same enzymes for different type of physiological regulation in different tissues. These various forms of enzymes, which catalyze same reaction, are called isozymes. The isozymes provide an avenue for varying regulation of the same reaction at distinct locations or times. Isozymes are homologous enzymes within a single organism that catalyze the same reaction but differ slightly in structure and more obviously in Km and Vmax values, as well as regulatory properties. Often, isozymes are expressed in a distinct tissue or organelle or at a distinct stage of development. For example hexokinase and are isozymes found in different tissues. Hexokinase has ubiquitous distribution and has low Km and high affinity for glucose pushes glucose in glycolysis and energy generation (catabolic reaction). However, when systemic glucose exceeds in the blood the glucokinase (found in liver), which has high Km and low affinity for glucose, phosphorylates it to deposit the excess glucose in the form of glycogen (anabolic reaction) in the liver.

Figure 17b: Regulation of enzyme activity by phosphorylation dephosphorylation (Covalent modification) at serine, threonine or tyrosine residue. 17.1.4 Reversible Post translational Modifications: The side chain of various amino acids in protein is susceptible to different type of covalent modifications after synthesis. Proteins may be phosphorylated, acetylated, methylated, sulfated, glycosylated, amidated, hydroxylated, prenylated, myristolated, often in a reversible fashion. The catalytic properties of many enzymes are markedly altered by the covalent attachment of a modifying group, most commonly a phosphoryl group on serine, threonine and tyrosine residues. Regulation by phosphorylation through the action of using ATP, and dephosphorylation by phosphates is extremely common (Figure 17b). Control of phosphorylation state is mediated through signal transduction process starting at the cell membrane, leading to the activation or inhibition of protein kinases and within the cell. Phosphorylation-dephosphorylation permits the functional properties of the affected enzyme to be altered only for as long as it serves a specific need. Once the need has passed, the enzyme can be converted back to its original form, poised to respond to the next stimulatory event. Another explanation underlying the widespread use of protein phosphorylation-dephosphorylation lies in the chemical properties of the phosphoryl group itself. In order to alter an enzyme’s functional properties (Figure 17c), any modification of its chemical structure must influence the protein’s three- dimensional configuration. The high charge density of protein-bound phosphoryl groups at physiologic pH and their propensity to form salt bridges with arginyl residues make them potent agents for modifying protein structure and function. Phosphorylation generally targets amino acids distant from the catalytic site itself. Consequent conformational changes then influence an enzyme’s intrinsic catalytic efficiency or other properties. Majority of cell signaling and metabolic reactions are predominantly controlled by phosphorylation and acetylation modifications.

Figure 17c: Phosphorylation of enzyme brings structural change to convert it into an active form, which can bind the substrate and catalyze the reaction.

The more common example of enzyme activation and deactivation by phosphorylation is glycogen , which is the first enzyme in degradation of glycogen to glucose. It exists in two forms called a and b (active and not active), which are phosphorylated and not phosphorylated. So the enzyme is regulated by the insertion or removal of a phosphate group on the enzyme. Histone proteins that assist in the packaging of DNA into chromosomes as well as in gene regulation are rapidly acetylated and deacetylated at lysine residues in vivo. More heavily acetylated histones are associated with genes that are being actively transcribed. The acetyltransferase and deacetylase enzymes are themselves regulated by phosphorylation, showing that the covalent modification of histones may be controlled by the covalent modification of the modifying enzymes. The half lives of proteins in cells are also controlled by protein acetylation. If proteins are acetylated at lysine the ubiquitin modification which also involves lysine cannot occur and therefore the half life of protein gets enhanced.

17.1.5 Proteolytic activation: Certain enzymes are synthesized as proenzymes (also called zymogens), which are inactive forms of enzymes that become active only after being cleaved at specific site in the polypeptide chain by specific (Figure 17d). A different regulatory motif is used to irreversibly convert an inactive enzyme into an active one. This regulatory mechanism generates digestive enzymes such as , , and . Caspases, which are proteolytic enzymes that are the executioners in programmed cell death (apoptosis) are proteolytically activated from the procaspase. The process of blood clotting is also a cascade of zymogen activations. Active digestive and clotting enzymes are switched off by the irreversible binding of specific inhibitory proteins.

Figure 17d: Several enzymes express in pro-enzyme form, when physiologically required, they are cleaved by proteases on definite site to obtain the active form and binding of the substrate.

Because cells lack the ability to reunite the two portions of a protein produced by hydrolysis of a peptide bond, constitutes an irreversible modification, unlike reversible covalent post translational modifications. The significance of control of enzyme through proteolytic regulation can be understood in this way that the synthesis and secretion of proteases as catalytically inactive proenzymes protects the tissue of origin (eg, the pancreas) from autodigestion. Certain physiologic processes such as digestion are intermittent but fairly regular and predictable. Others such as blood clot formation, clot dissolution, and tissue repair

are brought “on line” only in response to pressing physiologic or pathophysiologic need. The processes of blood clot formation and dissolution clearly must be temporally coordinated to achieve homeostasis. Enzymes needed intermittently but rapidly often are secreted in an initially inactive form since the secretion process or new synthesis of the required proteins might be insufficiently rapid for response to a pressing pathophysiologic demand such as the loss of blood.

17.1.6 Allosteric control: The activity of enzymes that catalyze key regulatory reactions of metabolic pathways are often subject to allosteric regulation. Jacques Monod proposed the existence of allosteric sites that are physically distinct from the catalytic site. Allosteric enzymes thus are those whose activity at the may be modulated by the presence of effectors at an allosteric site. This hypothesis has been confirmed by many lines of evidence, including x-ray crystallography and site-directed mutagenesis, demonstrating the existence of spatially distinct active and allosteric sites on a variety of enzymes. Primarily, the multi-subunit proteins/enzymes are Allosteric proteins/enzymes, which contain distinct regulatory sites and multiple functional sites. Such enzymes are generally controlled by allosteric control. Regulation by small signal molecules is a significant means of controlling the activity of many proteins. Such molecules which regulate the activity of allosteric enzymes are called the effector molecule. The binding of these regulatory molecules at allosteric sites (occupy another space) distinct from the active site triggers conformational changes that are transmitted to the active site, this structural change results in an apparent change in binding affinity of substrate (Figure 17e). This "action at a distance" through binding of one affecting the binding of another at a distinctly different site, is the essence of the allosteric concept. Moreover, allosteric proteins show the property of : activity at one functional site affects the activity at others. As a consequence, a slight change in substrate concentration can produce substantial changes in activity. It is not necessary that allosteric enzymes are oligomeric always, the property of allosteric regulation is observed in single polypeptide chain also. For example Glucokinase is a monomeric enzyme that displays a low affinity for glucose and a sigmoidal saturation curve for its substrate. This plays the role of a glucose sensor in pancreas and liver. However, allosteric enzymes are an exception to the Michaelis-Menten model. Because they have more than one active site, they do not obey the Michaelis-Menten kinetics but instead have sigmoidal kinetics (also see Module 16).

Figure 17e: Binding of effector molecule on allosteric site brings structural change in enzyme and converts it in to an active form facilitating the substrate binding and conversion in to product.

The enzymes which are displaying allosteric control are information transducers and their activity can be modified in response to signal molecules or to information shared among active sites. Therefore, allosteric enzymes play a crucial role in many fundamental biological processes like metabolic regulation and cell signaling. Allosteric enzymes are unique compared to other enzymes because of its ability to adapt various conditions in the environment due to its special properties. The binding of a nonsubstrate molecule to the allosteric site functions to influences the activity of the enzyme. Based on the positive and negative effector, the allosteric regulation of enzyme is called allosteric activation or allosteric inhibition respectively (Figure 17f). Allosteric activators bind to allosteric sites, inducing a conformational change that increases the affinity of the enzyme's active site for its substrate. However, In allosteric inhibition, inhibitor molecules bind to an enzyme at the allosteric site where their binding induces a conformational change that reduces the affinity of the enzyme's active site for its substrate.

Figure 17f: The model represents the allosteric activation and inhibition by positive and negative effector molecules respectively. In case of allosteric inhibition the high concentration of product itself or its metabolite acts as negative effector and inhibits the substrate binding.

The classical example of Allosteric control of enzyme is the binding of oxygen with hemoglobin. At low oxygen pressure the hemoglobin remains in T state (low oxygen binding), however increasing oxygen pressure brings change in tetrameric molecule which facilitates the complete saturation of hemoglobin with oxygen, R state. The binding of oxygen to hemoglobin is cooperative and regulated by various positive and negative effector molecules; beside the oxygen pressure itself, H+ and carbon dioxide concentration are other regulators. The 2,3 bisphosphoglycerate acts as negative effector of oxygen binding on hemoglobin and its binding on hemoglobin facilitates the dissociation of oxygen in tissues.

17.2 Feedback Inhibition: Majority of biochemical pathways contains several enzymatic reactions which are controlled by allosteric enzymes. Therefore, when the end product of any biochemical pathway inhibits the regulatory allosteric enzymes of the same pathway as negative effector then this phenomenon is called feedback inhibition. For example, conversion of X to Z through Y in a biochemical pathway is carried out by enzyme E1 and E2 (Figure 17g). When the concentration of Z increases in the cell, it acts as feedback inhibitor of E1 and minimizes or stops the rate of conversion of X into Y by inhibiting the enzyme E1. Cells use feedback inhibition to regulate enzyme activity mainly in metabolic pathways. Cells have evolved to use the products of their own reactions for feedback inhibition of enzyme activity. Metabolic reactions, such as

anabolic and catabolic processes, must proceed according to the demands of the cell. In order to maintain chemical equilibrium and meet the needs of the cell, some metabolic products inhibit the enzymes in the chemical pathway, while some reactants activate them.

Figure 17g: In a biochemical pathway consisting of two reactions the end product Z upon accumulation (high concentration) inhibits the first regulatory (allosteric) enzyme E1 to control the biochemical pathway.

The increase in the concentration of Z means the cell does not need the end product of the biochemical pathway anymore and therefore the end product starts accumulating. After the product has been utilized or broken down and its concentration thus decreased, the inhibition is relaxed, and the formation of the product resumes. Feedback inhibition is a mechanism by which the concentration of certain cell constituents is limited. Inhibition of E1 results not from the “backing up” of intermediates but from the ability of Z to bind and inhibit E1. Typically, Z binds at an allosteric site spatially distinct from the catalytic site of the target enzyme. Feedback inhibitors thus are allosteric effectors and typically bear little or no structural similarity to the substrates of the enzymes they inhibit. In this example (Figure 17.6), the feedback inhibitor Z acts as a negative allosteric effector of E1. The kinetics of feedback inhibition may be competitive, noncompetitive, partially competitive, or mixed. In a branched biochemical pathway, the initial reactions participate in the synthesis of several products. Feedback inhibitors, which frequently are the small molecule building blocks of macromolecules (eg, amino acids for proteins, nucleotides for nucleic acids), typically inhibit the first committed step in a particular biosynthetic sequence. A much-studied example is inhibition of bacterial aspartate transcarbamoylase by CTP. The enzyme catalyzes the first step in the synthesis of pyrimidines. The enzyme functions to catalyze the condensation of aspartate and carbamoyl phosphate to form carbamoylaspartate and orthophosphate. The enzyme ultimately catalyzes the reaction that will yield cytidine triphosphate (CTP). This allosteric enzyme is unique in that for high concentration of the final product CTP, the enzyme activity is low. However, for low concentrations of the final product CTP, the enzymatic activity is high. At high CTP concentration, CTP binds to the allosteric site. Thus, CTP functions as an allosteric inhibitor decreasing the enzymatic activity of the enzyme. This enzyme also has separate regulatory and catalytic subunits on separate polypeptide chains. There are instances though when CTP concentrations remain high and cells in the body need more enzyme. This is when a different allosteric molecule ATP functions to attach to the allosteric site and functions as enzyme activator enhancing the activity of the enzyme. Thus, even with high concentrations of

CTP, the enzyme activity could be enhanced because of ATP, which also acts on the allosteric site. This example explains the benefits of allosteric control and the ability of allosteric enzymes to adapt to various conditions of the environment. This is particularly helpful for cells because there are occasions when the cell requires an allosteric activator like "ATP" to enhance the enzyme even when it is inhibited due to high amounts of product. The production of both amino acids and nucleotides is controlled through feedback inhibition. ATP is the product of the catabolic of sugar (cellular respiration), but it also acts as an allosteric regulator for the same enzymes that produced it. ATP is an unstable molecule that can spontaneously dissociate into ADP; if too much ATP were present, most of it would go to waste. This feedback inhibition prevents the production of additional ATP if it is already abundant. While ATP is an inhibitor, ADP is an allosteric activator. When levels of ADP are high compared to ATP levels, ADP triggers the catabolism of sugar to produce more ATP. The lack of structural similarity between a feedback inhibitor and the substrate for the enzyme whose activity it regulates suggests that these effectors are not isosteric with a substrate but allosteric (“occupy another space”). Sometimes the end product dependent feed back regulation does not involve feed back inhibition. For example, while dietary cholesterol decreases hepatic synthesis of cholesterol, this feedback regulation does not involve feedback inhibition. HMG- CoA reductase, the rate-limiting enzyme of cholesterologenesis, is affected, but cholesterol does not feedback-inhibit its activity. Regulation in response to dietary cholesterol involves curtailment by cholesterol or a cholesterol metabolite of the expression of the gene that encodes HMG-CoA reductase.

17.3 Kinetic behaviour of allosteric enzymes: The biochemical reaction kinetics of those enzymes which have single active site follows Michaelis-Menten model. However, the same model does not apply for the kinetic properties of those enzymes which are allosteric in nature, because they bind their substrates in a cooperative fashion analogous to the binding of oxygen by haemoglobin. These enzymes consist of multiple subunits and/or multiple active sites per polypeptide chain with binding ability of substrates at different binding sites. In allosteric enzymes, the binding of substrate to one active site can affect the properties of other active sites in the same enzyme molecule. Due to this reason, the binding of substrate becomes Figure 17h: The graph shows the enzyme cooperative; that is, the binding of substrate to saturation curve of michaelis-menten and one active site of the enzyme facilitates sigmoidal curve of allosteric enzymes. substrate binding to the other active sites. Binding of the first substrate molecule at one site affects the binding of other substrate molecules in both positive and negative manner. For enzymes that display positive cooperativity in binding substrate usually display sigmoidal (because it resembles "S") curve, when the curve is plotted between the reaction velocity Vi and substrate concentration [S] (Figure 17h); as compared to the hyperbolic plots predicted by the Michaelis-Menten model. Hemoglobin and aspartate carbamoyltransferase (ATCase) are most studied models to study the kinetic properties of allosteric regulation.

The kinetic properties of allosteric enzymes are often explained in terms of a conformational change between a low-activity, low-affinity "tense" or T state and a high- activity, high-affinity "relaxed" or R state. These structurally distinct enzyme forms have been shown to exist in several known allosteric enzymes. Based on kinetic properties of allosteric enzymes, they can be sub-divided in two groups K-series and V-series enzymes. For K-series allosteric enzymes, the substrate saturation kinetics are competitive in the sense that Km is raised without an effect on Vmax. For V-series allosteric enzymes, the allosteric inhibitor lowers Vmax without affecting the Km. Alterations in Km or Vmax probably result from conformational changes at the catalytic site induced by binding of the allosteric effector at the allosteric site. For a K-series allosteric enzyme, this conformational change may weaken the bonds between substrate and substrate-binding residues. For a V-series allosteric enzyme, the primary effect may be to alter the orientation or charge of catalytic residues, lowering Vmax. Intermediate effects on Km and Vmax, however, may be observed consequent to these conformational changes. 17.3.1 Competitive and non-competitive inhibition: The cell uses specific molecules to regulate enzymes to either promote or inhibit certain chemical reactions. In competitive inhibition, an inhibitor molecule is similar enough to a substrate that it can bind to the active site and simply block the substrate from binding. In noncompetitive inhibition, an inhibitor molecule binds to the enzyme at a location other than the active site (an allosteric site). The substrate can still bind to the enzyme, but the inhibitior changes the shape of the enzyme so it is no longer in optimal position to catalyze the reaction. 17.4 Allosteric Interactions: An allosteric interaction occurs when the binding of a ligand to its site on a protein/receptor is able to modify the binding of another ligand to a topographically distinct site on the same receptor and vice versa. For example the muscarinic cholinoceptors represent the best-studied examples of allosteric phenomena among the G-protein-coupled receptor superfamily.

Orthosteric agonists bind to the -coupled receptor (GPCR), which induces a conformational change that results in the activation of downstream signalling. Positive allosteric modulators are allosteric ligands that bind to a topographically distinct site to the orthosteric agonist and enhance the affinity (cooperativity factor-α) and/or efficacy (modulation factor-β) of the orthosteric agonist. Negative allosteric modulators are allosteric ligands that decrease the affinity (cooperativity factor-α) and/or efficacy (modulation factor-β) of the orthosteric agonist. Allosteric ligands that have no effect on the affinity and/or efficacy mediated by the orthosteric agonist are termed neutral allosteric ligands. Allosteric modulators offer a number of potential therapeutic advantages, including a ceiling level to their effects and the possibility of 'absolute selectivity' of action, based on the degree of co-operativity rather than the affinity of the modulator for any one receptor subtype. Allosteric interaction of ligands with protein can be of homotropic type when the same molecule binds on one site and affects the binding of other similar molecules on other sites like oxygen binding on hemoglobin or heterotropic type when the binding of positive effector molecule on allosteric site facilitates the binding of substrate molecule on active site.

17.5 Summary

 The activity of proteins, including enzymes, often must be regulated so that they function at the proper time and place. The control of enzyme activity is essential in maintaining the steady state of all organisms.  The substrates for most enzymes are usually present at a concentration close to Km. This facilitates passive control of the rates of product formation response to changes in levels of metabolic intermediates.  The enzymatic reactions are predominantly regulated by the concentration of substrate, enzyme turnover, different forms of enzymes called isozymes, reversible co-valent modifications, proteolytic activation and most important allostery.  Protein phosphorylation and de-phosphorylation is the major regulator of enzyme regulation by co-valent modification.  Phosphorylation by protein kinases at serine, threonine and tyrosine residues and subsequent dephosphorylation by protein phosphatises regulates the activity of many human enzymes. The protein kinases and phosphatases that participate in regulatory cascades which respond to hormonal or second messenger signals integrate complex environmental information to produce an appropriate and comprehensive cellular response.  Selective proteolysis of catalytically inactive proenzymes initiates conformational changes that form the active site. Secretion as an inactive proenzyme facilitates rapid mobilization of activity in response to injury or physiologic need and may protect the tissue of origin (eg, autodigestion by proteases).  Allostery is the major regulator of enzyme activity in the cells  Binding of metabolites and second messengers to allosteric sites distinct from the catalytic site of enzymes triggers conformational changes that alter Vmax or the Km.  The allostric effectors can be of two types, the effectors which upon binding facilitates the binding of substrate is called positive effector however when the effector inhibits the binding of substrate on the enzyme, it is called negative effector molecule.  When the high concentration of final product of any biochemical pathway inhibits the regulatory enzymes of that pathway, it is called feedback inhibition. Most of the metabolic pathways in the cells are regulated through feedback inhibition.