INTRODUCTION – REGULATION OF ENZYME ACTION
- The activity of enzymes, often must be regulated so that they function at the proper time and place. The biological activity of enzyme is regulated in four principal ways:
- Allosteric control. Allosteric enzyme contain distinct regulatory sites and multiple functional sites. Regulation by small signal molecules is a significant means of controlling the activity of many proteins. The binding of these regulatory molecules at sites distinct from the active site triggers conformational changes that are transmitted to the active site. Moreover, allosteric proteins enzyme show the property of cooperativity: 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
2. Multiple forms of enzymes :- Isozymes, or isoenzymes, 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.
3. Reversible covalent modification :- The catalytic properties of many enzymes are markedly altered by the covalent attachment of a modifying group, most commonly a phosphoryl group. ATP serves as the phosphoryl donor in these reactions, which are catalyzed by protein kinases. The removal of phosphoryl groups by hydrolysis is catalyzed by protein phosphatases.
4. Proteolytic activation :- The enzymes controlled by some of these mechanisms cycle between active and inactive states. A different regulatory motif is used to irreversibly convert an inactive enzyme into an active one. Many enzymes are activated by the hydrolysis of a few or even one peptide bond in inactive precursors called zymogens or proenzymes. This regulatory mechanism generates digestive enzymes such as chymotrypsin, trypsin, and pepsin. Caspases, which are proteolytic enzymes that are the executioners in programmed cell death, or apoptosis are proteolytically activated from the procaspase form. Blood clotting is due to a remarkable cascade of zymogen activations. Active digestive and clotting enzymes are switched off by the irreversible binding of specific inhibitory proteins.
Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines, bases that are components of nucleic acids. The reaction catalyzed by this enzyme is the condensation of aspartate and carbamoyl phosphate to form N-carbamoylaspartate and orthophosphate.
ATCase catalyzes the committed step in the pathway that will ultimately yield pyrimidine nucleotides such as cytidine triphosphate (CTP). How is this enzyme regulated to generate precisely the amount of CTP needed by the cell?
John Gerhart and Arthur Pardee found that ATCase is inhibited by CTP, the final product of the ATCase-controlled pathway. The rate of the reaction catalyzed by ATCase is fast in the absence of high concentrations of CTP but decreases as the CTP concentration increases. Thus, more molecules are sent along the pathway to make new pyrimidines until sufficient quantities of CTP have accumulated.
The effect of CTP on the enzyme exemplifies the feedback, or end-product, inhibition. Despite the fact that end-product regulation makes considerable physiological sense, the observation that ATCase is inhibited by CTP is remarkable because CTP is structurally quite different from the substrates of the reaction. Owing to this structural dissimilarity, CTP must bind to a site distinct from the active site where substrate binds. Such sites are called allosteric (from the Greek allos, “other,” and stereos, “structure”) or regulatory sites. CTP is an example of an allosteric inhibitor. In ATCase (but not all allosterically regulated enzymes), the catalytic sites and the regulatory sites are on separate polypeptide chains.
ATCase can be literally separated into regulatory and catalytic subunits by treatment with a mercurial compound such as p-hydroxymercuribenzoate, which reacts with sulfhydryl groups.
MULTIPLE FORMS OF AN ENZYME
Isozymes or isoenzymes, are enzymes that differ in amino acid sequence yet catalyze the same reaction. Isozymes can often be distinguished from one another by biochemical properties such as electrophoretic mobility.
The existence of isozymes permits the fine-tuning of metabolism to meet the particular needs of a given tissue or developmental stage. Consider the example of lactate dehydrogenase (LDH), an enzyme that functions in anaerobic glucose metabolism and glucose synthesis. Human beings have two isozymic polypeptide chains for this enzyme: the H isozyme highly expressed in heart and the M isozyme found in skeletal muscle.
The amino acid sequences are 75% identical. The functional enzyme is tetrameric, and many different combinations of the two subunits are possible. The H4 isozyme, found in the heart, has a higher affinity for substrates than does the M4 isozyme. The two isozymes also differ in that high levels of pyruvate allosterically inhibit the H4 but not the M4 isozyme. The other combinations, such as H3M, have intermediate properties depending on the ratio of the two kinds of chains.
REVERSIBLE COVALENT MODIFICATION
An important example of enzyme regulation by phosphorylation is the case of glycogen phosphorylase (Mr 94,500) of muscle and liver which catalyzes the reaction The glucose 1-phosphate so formed can be used for ATP synthesis in muscle or converted to free glucose in the liver.
Note that glycogen phosphorylase, though it adds a phosphate to a substrate, is not itself a kinase, because it does not utilize ATP or any other nucleotide triphosphate as a phosphoryl donor in its catalyzed reaction. It is, however, the substrate for a protein kinase that phosphorylates it.
Glycogen phosphorylase occurs in two forms: the more active phosphorylase a and the less active phosphorylase b. Phosphorylase a has two subunits, each with a specific Ser residue that is phosphorylated at its hydroxyl group. These serine phosphate residues are required for maximal activity of the enzyme. The phosphoryl groups can be hydrolytically removed by a separate enzyme called phosphoprotein phosphatase 1 (PP1)
Phosphorylase b can be reconverted (reactivated) to phosphorylase a by the action of phosphorylase kinase. In this reaction, phosphorylase a is converted to phosphorylase b by the cleavage of two serine phosphate covalent bonds, one on each subunit of glycogen phosphorylase. Phosphorylase b can, in turn, be reactivated—covalently transformed back into active phosphorylase a—by another enzyme, phosphorylase kinase, which catalyzes the transfer of phosphoryl groups from ATP to the hydroxyl groups of the two specific Ser residues in phosphorylase b: The breakdown of glycogen in skeletal muscles and the liver is regulated by varying the ratio of the two forms of glycogen phosphorylase.
The a and b forms differ in their secondary, tertiary, and quaternary structures; the active site undergoes changes in structure and, consequently, changes in catalytic activity as the two forms are interconverted.
The regulation of glycogen phosphorylase by phosphorylation illustrates the effects on both structure and catalytic activity of adding a phosphoryl group. In the unphosphorylated state, each subunit of this enzyme is folded so as to bring the 20 residues at its amino terminus, including some basic residues, into a region containing several acidic amino acids; this produces an electrostatic interaction that stabilizes the conformation.
Phosphorylation of Ser 14 interferes with this interaction, forcing the amino-terminal domain out of the acidic environment and into a conformation that allows interaction between the P -Ser and several Arg side chains. In this conformation, the enzyme is much more active.
Phosphorylation of an enzyme can affect catalysis in another way: by altering substrate-binding affinity. For example, when isocitrate dehydrogenase (an enzyme of the citric acid cycle; is phosphorylated, electrostatic repulsion by the phosphoryl group inhibits the binding of citrate (a tricarboxylic acid) at the active site.
In a regulatory cascade, a signal leads to the activation of protein X. Protein X catalyzes the activation of protein Y. Protein Y catalyzes the activation of protein Z, and so on. Since proteins X, Y, and Z are catalysts and activate multiple copies of the next protein in the chain, the signal is amplified in each step.
Fibrinogen is a dimer of heterotrimers (Aα2Bβ2γ2 ) and thereby activated for blood clotting, by the proteolytic removal of 16 amino acid residues from the amino-terminal end (the A peptide) of each α subunit and 14 amino acid residues from the aminoterminal end (the B peptide) of each β subunit. Peptide removal is catalyzed by the serine protease thrombin.
The fundamental reaction in the clotting of blood is the conversion of soluble plasma protein into insoluble fibrin. This conversion is catalyzed by the enzyme thrombin at the site of injury. Prothrombinase convert prothrombin into thrombin.
The clotting cascade may be triggered by the extrinsic pathway or intrinsic pathway.
The extrinsic pathway of blood clotting is shorter pathway and occur rapidly. It is so named because a tissue protein called tissue factor (also known as tissue thromboplastin or factor 3), leak into the blood from the cell outside (extrinsic to) blood vessels. In the presences of calcium, thromboplastin begins a sequence of reaction that ultimately activates clotting factor X.
The intrinsic pathway of blood clotting is more complex than extrinsic pathway, and it occur more slowly. The intrinsic pathway is so named because its activators are either in direct contact with blood or contain within ( intrinsic to) the blood outside tissue damage is not needed. If endothelial cells become damaged, blood can come in contact with collagen fibers in the connective tissue around the endothelium of the blood vessels. Contact with collagen fibers activates clotting factor XII which begins sequence of reaction that eventually activates clotting factor X.
- The activities of metabolic pathways in cells are regulated by control of the activities of certain enzymes.
- The activity of an allosteric enzyme is adjusted by reversible binding of a specific modulator to a regulatory site. A modulator may be the substrate itself or some other metabolite, and the effect of the modulator may be inhibitory or stimulatory. The kinetic behavior of allosteric enzymes reflects cooperative interactions among enzyme subunits.
- Other regulatory enzymes are modulated by covalent modification of a specific functional group necessary for activity. The phosphorylation of specific amino acid residues is a particularly common way to regulate enzyme activity.
- Many proteolytic enzymes are synthesized as inactive precursors called zymogens, which are activated by cleavage to release small peptide fragments.
- Blood clotting is mediated by two interlinked regulatory cascades of proteolytically activated zymogens.
- Enzymes at important metabolic intersections may be regulated by complex combinations of effectors, allowing coordination of the activities of interconnected pathways.
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