Enzyme Activity & Regulation PDF

# Quantitative assay of enzymes catalytic activity - The amount of an enzyme in a solution or tissue extract can be assayed in terms of the catalytic effect it produces. - In an enzyme-catalyzed reaction, the catalytic activity of the enzyme is reflected by the rate at which the substrate is transf...

# Quantitative assay of enzymes catalytic activity - The amount of an enzyme in a solution or tissue extract can be assayed in terms of the catalytic effect it produces. - In an enzyme-catalyzed reaction, the catalytic activity of the enzyme is reflected by the rate at which the substrate is transformed to the product. - Greater the rate of transformation of substrate to product, more the enzyme activity and vice versa. - Rate of transformation may be estimated from the rate of appearance of product and/or the rate of disappearance of the substrate. # Measurement of Activity - The activity of the enzyme is measured in terms of the following: - One-unit enzyme activity is defined as the amount causing transformation of 1.0 mmole of substrate per minute at 25°C, under optimum conditions of measurement. - It is usually expressed as mmole of substrate disappeared, or mmole of product formed per minute (mmole = 10^-3moles). # Turnover number of enzyme - This refers to the number of substrate molecules transformed per unit time by a single enzyme molecule (or by a single catalytic site), when the enzyme concentration alone is the rate-limiting factor. - For example, carbonic anhydrase has a turnover number of 36,000,000. | Enzyme | Turnover number | |--------------------|-------------------| | Carbonic anhydrase | 36,000,000 | | B-Amylase | 11,00,000 | | Phosphoglucomutase | 1,240 | | Fumarase | 800 | # Reaction activation energy - All chemical reactions require some amount of energy to get them started or it is first push to start reaction. This energy is called activation energy. - Enzymes lower the reaction activation energy. # The Activation Energy Barrier - Every chemical reaction between molecules involves bond breaking and bond forming. - The initial energy needed to start a chemical reaction is called the activation energy ($E_A$). - Activation energy is often supplied in the form of thermal energy that the reactant molecules absorb from their surroundings. - Enzymes catalyze reactions by lowering the $E_A$ barrier. - Enzymes do not affect the change in free energy (ΔG). # Energy changes occurring during the reaction - Virtually all chemical reactions have an energy barrier separating the reactants and the products. This barrier, called the free energy of activation. - Free energy is a useful thermodynamic function for understanding enzyme-catalyzed reactions. - The free energy of a reaction is the difference between its reactants and its products, this called the ΔG. If ΔG is negative, the reaction will occur spontaneously, "exergonic." If ΔG is positive, energy input is required, "endergonic." # Exergonic and Endergonic Reactions - An exergonic reaction proceeds with a net release of free energy and is spontaneous. - An endergonic reaction absorbs free energy from its surroundings and is nonspontaneous. # ATP powers cellular work by coupling exergonic reactions to endergonic reactions - To do work, cells manage energy resources by energy coupling, the use of an exergonic process to drive an endergonic one. Overall, the coupled reactions are exergonic. - Most energy coupling in cells is mediated by ATP. # Structure of ATP and its hydrolysis A diagram of ATP and its hydrolysis into ADP, inorganic phosphate, and energy is described in the document. # Mechanisms for Regulation of Enzyme Activity - Regulation of enzymes activities is essential for coordinating numerous metabolic processes and altering the rate of a pathway according to the overall metabolic needs of the body. - There are 3 major mechanisms for regulating the enzyme activity: - Allosteric modulation. - Covalent modification. - Induction-repression of enzyme synthesis. # A-Allosteric Modulation - Some enzymes, called allosteric enzymes, exist in alternate higher order structures. - The equilibrium between these alternative structural forms can be affected by binding of regulatory ligands, termed allosteric modulators. - The binding of a ligand can either enhance the activity of the enzyme (allosteric stimulation) or inhibit it (allosteric inhibition). - The allosteric enzymes possess a site distinct and physically separate from the substrate-binding site; it is known as the allosteric site. - The term allosteric is derived from the Greek word, (allo) which means other, and (steric) means space or site. # Allosteric modulators bind with the allosteric site by reversible non-covalent interactions. A diagram of an allosteric enzyme with an active site, allosteric site, allosteric effector, and substrate bound to the active site is described in the document. - This binding alters conformation of the enzyme, which in turn leads to alteration of the enzyme activity thereby providing important means of its regulation. - The allosteric site is specific for the modulator, just as the active site is for the substrate. - Some modulators, called the allosteric activators (positive modulators), increase the enzyme activity in this manner, whereas others, called the allosteric inhibitors (negative modulators), decrease the enzyme activity. # B-Covalent Modification - There are two general types of covalent modifications of enzymes that regulate their activities. - Reversible interconversion of active and inactive states of an enzyme by covalent attachment of specific group(s). - Irreversible activation of inactive enzyme precursor by proteolytic cleavage. # i. Reversible Covalent Regulation - Activities of several enzymes are regulated by covalent attachment of specific groups, such as phosphate, calcium or nucleotide. - This results in altered charged-state of the enzyme molecule, which in turn causes change in shape of the enzyme and hence its activity. # Example on Reversible Covalent Regulation - Phosphorylation - dephosphorylation A diagram of phosphorylation and dephosphorylation of an enzyme is described in the document. #- R groups of some amino acids present at the active site of enzymes carry hydroxyl group (e.g. threonine, serine and tyrosine), which can be phosphorylated by protein kinase. - This enzyme causes attachment of a phosphate group obtained from ATP to hydroxyl group(s) of these amino acid(s). - This puts a large negative charge in this region of the protein, which causes it to change its shape and its activity. # ii. Irreversible Activation by Proteolytic Cleavage - Several proteins are synthesized in inactive form. These are called Zymogens, e.g. protein digesting enzymes and blood clotting proteins. - They are activated when a small length of the protein is cleaved off from one end through action of specific proteases. This causes an irreversible rearrangement of the tertiary structure to yield the active form of the protein. # C-Induction-repression of Enzyme Synthesis - The allosteric modulation and the covalent modifications change the activity of the regulatory enzymes. In addition, there exist another mechanism that operates at gene-level to regulate the enzyme synthesis or breakdown. - This may cause increased synthesis (Induction) or decreased synthesis (repression) of the enzyme protein and hence its intracellular concentration also changes accordingly. # Examples: - The sex hormones estrogen and testosterone are two very important regulators of some enzymes' genes. - Insulin induces some glycolytic enzymes and represses some enzymes of gluconeogenesis. - Alterations in enzyme levels as a result of induction or repression of protein synthesis are slow (hours to days), compared with allosterically or covalently regulated changes in enzyme activity, which occur in seconds to minutes. # Isoenzymes - These are closely related enzyme proteins that catalyze the same reaction but differ in their: - molecular structure, kinetic parameters, sensitivity to inhibitors or response to allosteric modulators. - They may act on the same substrate, but with different K_m and V_max values. # Mechanisms for production of isoenzymes 1. Different combinations of polypeptide subunits to form an active polymeric enzyme. These subunits arise from different genetic loci; This is the most common mechanism for formation of isoenzymes. - For example, - Creatine kinase (CK) occurs as a dimer with two types of subunits: M (muscle) and B (brain), which are products of loci in chromosomes 14 and 19, respectively. Three combinations of these subunits are MM, MB, and BB. - Lactate dehydrogenase occurs as a tetramer with two types of polypeptide subunits (H and M) that are arranged differently to yield five isoenzymic forms, LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5, are made up of different ratios of LDH-M and LDH-H subunits. A diagram of the different subunits of LDH is described in the document. 2. Some isoenzymes, called allelic isozymes (allozymes) arise from the same genetic locus, and yet they exhibit structural variations among different individuals. They are referred to as different alleles or alternate forms of the same enzyme. - For example, about 400 different allelic isoenzymes of glucose 6-phosphate dehydrogenase have been identified; only one form is present in a certain individual, but all different forms are seen in the whole population. 3. Post-translational modification of the same enzyme polypeptide may occur in different ways to generate different isoenzyme, called iso-forms. by covalently adding functional groups such as phosphate, acetate, amide, or methyl groups to proteins. As a result, Post-translational modified enzymes such as kinases and glycosyltransferases are targets or are starting to be targeted for drug development. - Several isoforms of the enzyme alkaline phosphatase are known, differing in the number of sialic acid residues, and therefore, the number of the charged group. # Separation of Isoenzymes 1. Electrophoresis - Different subunit compositions or primary structures yield different ionic charges to the polypeptide chains - this permits their separation by electrophoresis. - Several isoenzyme studies have relied upon different electrophoretic migration patterns. 2. Use of inhibitors - High temperature or some inhibitor may inhibit an isoenzyme, which forms the basis for its identification. - For example, - Alkaline phosphatase (ALP) has 4 isoenzymes; Intestinal ALP, Placental ALP, Germ cell ALP and tissue nonspecific ALP or liver/bone/kidney (L/B/K) ALP. - Placental ALP is heat stable; even at 65°C but (L/B/K) ALP loses its activity at this temperature in 30 minutes. - Intestinal ALP is inhibited by phenylalanine. # Enzymes in Clinical Diagnosis - Enzyme assays are performed in blood samples and other body fluids to diagnose a wide range of clinical conditions. - Tissue localization of some important enzymes: | Enzyme | Main localization | |------------------------------------|------------------------------------------------------------------------| | Aspartate transaminase | Heart, liver, skeletal muscle, kidney, brain | | Alanine transaminase | Liver, skeletal muscle, kidney, brain | | Alkaline phosphatase | Bone (osteoblasts), intestinal mucosa, liver, placenta, saliva | | Acid phosphatase | Prostate, erythrocyte | | Lactate dehydrogenase | Heart, liver, skeletal muscle, kidney, erythrocytes, pancreas, lung | | y- Glutamyl transpeptidase | Liver | | Creatine Kinase | Skeletal muscle, heart, brain | | α-Amylase | Pancreas, saliva | | Aldolase | Skeletal muscle, heart | | Arginase | Liver | | Acetylcholinesterase | Brain, nervous tissue, erythrocytes | | 5'-Nucleotidase | Hepatobiliary tract, pancreas | | Glucose 6-phosphatase | Liver | - Cellular enzymes are usually restricted in the cells at which they are synthesized and where they function. - During normal cellular turnover they are released into plasma, from where they are subsequently removed and eliminated from the body. - In plasma they do not perform a physiological function and their plasma levels in healthy state are low to absent. - Under pathological conditions, the serum levels of cellular enzymes can increase, because disease process can increase the cell-membrane permeability and release of intracellular enzymes into serum. - Such as: - Metabolic stress without necrosis can also lead to elevated enzyme levels, apparently by a transient increase of the membrane permeability. - Neoplastic diseases also result in elevated enzyme levels when the tumor invades and destroys tissues. - Each cell type has its characteristic complement of enzymes and so identification of the elevated enzymes in plasma can point towards the damage of particular tissues and indicates the severity of the disease and the prognosis for the patient. - For example, - Following a block in coronary blood vessel, oxygen deprivation damages the heart muscles and activities of the myocardial enzymes, e.g. LDH and CK in blood rise markedly. - Alcohol dehydrogenase is unique to liver. - Acid phosphatase is established clinical use in diagnosis of metastatic prostatic carcinoma. # Clinical enzymology is a useful diagnostic tool and several enzymes are estimated routinely in most clinical laboratories. - Commonly assayed enzymes for specific diagnosis: | Enzyme | Diseases | |------------------------------------|-----------------------------------------------------------------------------------------------------------------------------------| | Aspartate transaminase (AST) | Rises in myocardial infarction after CK and returns to normal in 4-5 days. Early indicator of hepatocellular damage. | | Alanine transaminase (ALT) | Marked elevation in acute hepatitis (viral or toxic) and in other parenchymal liver disease. | | Alkaline phosphatase (ALP) | Marked elevation in obstructive liver disease and in bone diseases with increased osteoblastic activity, e.g. rickets. | | Acid phosphatase (ACP) | Marker for carcinoma prostate. Rises in metastatic bone diseases, especially from primary from prostate. | | Lactate dehydrogenase (LDH) | Rises in myocardial infarctions after CK and AST; LDH, becomes more than LDH, (called flipped pattern). | | y-Glutamyl transpeptidase | Liver disease, especially alcoholism. | | Creatine Kinase (CK) | Marked increase in muscle disease (CK-MM) and in myocardial infarction (CK-MB is the first enzyme to rise). | | Amylase | About 1000-fold rise in acute pancreatitis. | | Lipase | Highly elevated in acute pancreatitis. | | Prostate specific antigen | Marker for carcinoma prostate. | - LDH, and LDH₂ = lactate dehydrogenase isoenzymes 1 and 2. # Transaminases - AST or GOT and ALT or GPT are two important transaminases, providing important diagnostic clues in hepatic, cardiac and skeleto-muscular disorders. - Other conditions characterized by increased plasma AST include acute pancreatitis, degenerative diseases of skeletal muscles, severe hemolytic anemia. # Phosphatases - ALP catalyzes hydrolytic removal of phosphate group from organic phosphate esters at an alkaline pH 9. A diagram describing how ALP catalyzes the hydrolytic removal of phosphate from a phosphate monoester is shown in the document. # Distribution and diagnostic utility - Highest concentration of ALP is found in liver, bone, intestine, and placenta. It is especially useful in diagnosis of bone and liver pathology, since these are the major sources of the enzyme. - Malignant disorders, primary or metastatic, may raise the enzyme activity because of two factors: - Some malignant cells can produce ALP. - Secondary infiltration of bone or liver by malignant cells cause damage to these organs. # Acid Phosphatase (ACP) - Acid phosphatase also catalyzes the hydrolysis of phosphate from a variety of phosphate esters. It is so named because it exhibits an acid pH 5–6. # Diagnostic utility - Elevation of acid phosphatase occurs with metastatic carcinoma of prostate. Normal prostate does not release significant acid phosphatase into the circulation, whereas metastatic prostate tissue does so. # Dehydrogenases # Lactate Dehydrogenase (LDH) - It is an enzyme of anaerobic glycolysis that catalyzes the reversible interconversion of lactate and pyruvate. A diagram of the process of LDH catalyzing the interconversion of pyruvate and lactate, including NAD⁺ and NADH, is shown in the document. # Distribution and diagnostic utility - It is widely distributed in all human tissues; therefore, its plasma level is elevated in a wide variety of diseases, including liver diseases and myocardial infarction, or hemolytic anemia when erythrocytes are degraded more rapidly than normally. - Fortunately, different tissues contain different isoenzymes, which enhance its diagnostic utility. # Isoenzymes of LDH - Isoenzymes are formed from two different subunits, H and M, which have essentially the same molecular weight (34,000) but different charge patterns. - Tissue distribution of lactate dehydrogenase isoenzymes: | Isoenzymes | Subunit composition | Tissue | Percentage in serum | |------------|-----------------------|--------------------------------|----------------------| | LDH₁ | HHHH | Myocardium, RBC | 30 | | LDH₂ | HHHM | Myocardium, RBC | 35 | | LDH₃ | HHMM | Brain, kidney | 20 | | LDH₄ | HMMM | Skeletal muscle, liver | 10 | | LDH₅ | MMMM | Skeletal muscle, liver | 5 | - They combine in different combinations to yield five isoenzymes which can be separated by electrophoresis. - The isoenzyme 1, a tetramer of four H subunits moves fastest at pH 8.6, and the isoenzyme 5, consisting of four M subunits, moves slowest at the same pH. # Tissue distribution of LDH - The isoenzyme pattern of a given tissue depends on the relative amounts of H and M subunits produced by its cells, which differs markedly among different tissues. - Thus, liver and skeletal muscles produce mostly M subunits; myocardium and bone marrow produce mostly H subunits; and most other tissues (lung, brain, kidney, pancreas) produce both. - Thus LDH1 (HHHH) is the predominant isoenzyme in cardiac muscle and erythrocytes. Therefore, in myocardial infarction and hemolytic anemia, the LDH1 is raised. - LDH4 and LDH5 are predominantly elevated in muscle trauma and in liver disorders. # β-Hydroxybutyrate Dehydrogenase - β-Hydroxybutyrate dehydrogenase catalyzes the oxidation of β-hydroxybutyrate by NAD⁺ to yield acetoacetate and NADH. A diagram of the oxidation of β-hydroxybutyrate by NAD⁺ catalyzed by β-hydroxybutyrate dehydrogenase is shown in the document. - The enzyme has a wide distribution, being present in most human cells. Its activity is elevated following a myocardial infarction. # Creatine Kinase (CK) - It is an important enzyme in energy metabolism that catalyzes transfer of the phosphate group of creatine phosphate to ADP producing ATP, thereby serving as an immediate source of ATP in contracting muscles. A diagram of the process of CK catalyzing the transfer of the phosphate group of creatine phosphate to ADP is shown in the document. - It is a reasonably specific muscle enzyme, found in heart and skeletal muscle. Its activity rises in some diseases related to muscular dystrophy.. # Isoenzymes of CK - The isoenzymes are tissue specific and are very useful for the specific diagnosis of myocardial infarction. - Two slightly different gene products contained in the muscle (M) and the brain (B) correspond to CK. - The active CK is a dimer, and three isoenzyme forms are therefore possible: CK1 (BB), CK2 (MB) and CK3 (MM). - The numbering is based on electrophoretic mobility; CK1 has the greatest anodic mobility at pH 8.6. CK1 occurs in brain, CK2 occurs in heart and CK3 in skeletal muscles. # Troponin T and Troponin I are regulatory proteins involved in myocardial contractility. - They are released into the plasma in response to cardiac damage. # Cardiac troponin I (cTnI) is highly sensitive and specific for damage to cardiac tissue. - cTnI appears in plasma within 4-6 hours after an MI, peaks in 8-28 hours, and remain elevated for 3–10 days. # Elevated serum troponin are more predictive of adverse outcomes in myocardial infarction than the conventional assay of CK2. # Amylase and Lipase - These are the digestive enzymes secreted by exocrine pancreas, and their serum levels are most notably increased in acute pancreatitis. - Though most pronounced elevations occur in acute pancreatitis, modest elevations of these enzymes may occur in various extra pancreatic disorders; - Amylase levels are elevated in inflammation of salivary glands such as that produced by mumps, and - lipase levels are elevated in intestinal infarction. # Enzymes as Therapeutic Agents - Enzymes find widespread use in treatment of several diseases such as: - Streptokinase or Urokinase can cause lysis of intravascular clots and therefore used in myocardial infarction. - Asparaginase is used as an anticancer drug; most commonly in some types of leukemia. - Pepsin and trypsin are useful in patients with chronic indigestion and in pancreatic insufficiency. - Alpha-1-antitrypsin (AAT) is used in treatment of a type of emphysema that is caused by deficiency of AAT. - Collagenase degrades collagenous tissue, and so used in management of severe burns and dermal ulcers. - Lysozyme (endogenous antibiotic) is found in human tears. It has antibacterial properties being active against cellulose of bacteria and is used in the infection of eye. - Penicillinase is a bacterial enzyme which degrades penicillin. Therefore, it finds use in management of patients who are allergic to penicillin.