Enzymes Biochemistry for Nursing Summer semester 2015 Dr

Enzymes Biochemistry for Nursing Summer semester, 2015 Dr. Mamoun Ahram

What are enzymes? • Enzymes are specialized proteins that are able to conduct chemical reactions under biological conditions. • Most enzymes have very specific functions, and convert specific substrates to the corresponding products.

Enzymes are catalysts • Enzymes are catalysts. – They increase the rate of a reaction. – Enzymes accelerate reactions by factors of as much as a million or more. • They are used in small amounts relative to the reactants. • They are not consumed in the reaction. • Enzymes do not change an energetically unfavorable into a favorable one.

How to express an enzymatic reaction? • Reactants are known as substrates. • We can express an enzymatic reaction like this: E + S ES EP E + P where E is the free enzyme; S is the free substrate, ES is the enzyme-substrate complex; P is the product of the reaction; and EP is the enzyme-product complex before the product is released • To simplify the reaction, the reaction is expressed as E + S ES E + P

Turnover number • A simple reaction of the hydration of carbon dioxide is catalyzed by an enzyme called carbonic anhydrase. • Each enzyme molecule can hydrate 106 molecules of CO 2 per second. • The catalyzed reaction is 107 times as fast as the uncatalyzed one. • The catalytic activity of an enzyme is measured by its turnover number, the maximum number of substrate molecules converted by one molecule of enzyme per unit time.

General properties of enzymes • The function of nearly all proteins depends on their ability to bind other molecules (ligands). • Two properties of a protein in regards to its interaction with ligands: – affinity: the strength of binding between a protein and other molecules – specificity: the ability of a protein to bind one molecule in preference to other molecules

Examples of very specific enzymes Proteases

Some enzymes are less specific Carbxypeptidase

Active sites of enzymes • Each enzyme has a specific three-dimensional shape that includes a region where the biochemical reaction takes place, called the active site • The active site contains a specialized amino acid sequence that facilitates the reaction.

Features of active site 1 • Binding is specific due to the precise interaction of the substrate with the enzyme. • Binding occurs at least at three points. • This illustrates the importance of chirality. • If a substrate is chiral, an enzyme usually catalyzes the reaction of only one of the pair of enantiomers because only one fits the active site in such a way that the reaction can occur.

Features of active site 2 • The active site is small. • The “extra” amino acids: – create threedimensional structure of active site, – form regulatory sites that interact with other proteins or small molecules and control the activity of the enzyme.

Features of active site 3 • Active sites are structures that look like canals that bind substrate molecules.

Features of active site 4 • Substrates are bound to enzymes by multiple weak attractions including : – – electrostatic interactions, hydrogen bonds, van der Waals forces, and hydrophobic interactions.

Naming of enzymes • In general, enzymes end with the suffix (-ase) • Most other enzymes are named for their substrates and for the type of reactions they catalyze, with the suffix “ase” added – An ATPase is an enzyme that breaks down ATP – ATP synthase is an enzyme that synthesizes ATP • Some enzymes have common names that provide little information about the reactions that they catalyze • Examples include the proteolytic enzyme trypsin

Classification of enzymes • Enzymes were classified into six major groups – Oxidoreductases – Transferases – Hydrolases – Lyases – Isomerases – Ligases

Oxidoreductases • • These enzymes catalyze oxidation and reduction reactions involving the transfer of hydrogen atoms or electrons This group can be further divided into 4 main classes: – – Dehydrogenases Oxidases Peroxidases Oxygenases

Dehydrogenases • • • Dehydrogenases catalyze Removal of 2 hydrogens to form a double bond. Usually, hydrogen is transferred from the substrate to a molecule known as nicotinamide adenine dinucleotide (NAD+) Lactate dehydrogenase catalyzes this reaction

Alcohol dehydrogenase • Another example is alcohol dehydrogenase

Oxidases • • Oxidases catalyze hydrogen transfer from the substrate to molecular oxygen producing hydrogen peroxide (H 2 O 2) as a one product (or addition of O 2 to a substrate) Glucose oxidase catalyzes this reaction: -D-glucose + O 2 gluconolactone + H 2 O 2

Peroxidases • • Peroxidases catalyze oxidation of a substrate by hydrogen peroxide. Example: the oxidation of two molecules of glutathione (GSH) in the presence of hydrogen peroxide:

Oxygenases • • • Oxygenases catalyze substrate oxidation by oxygen The reduced product of the reaction in this case is water and not hydrogen peroxide An example of this is the oxidation of lactate to acetate catalyzed by lactate-2 -monooxygenase CH 3 -CH(OH)-COOH + O 2 �CH 3 COOH + CO 2 + H 2 O

Transferases • These enzymes transfer a functional group (C, N, P or S) from one substrate to an acceptor molecule • Types: transaminases and kinases

Transaminases • A transaminase transfers an amino functional group from one amino acid to a keto acid, converting the amino acid to a keto acid and the keto acid to an amino acid • This allows for the interconversion of certain amino acids

Kinases transfer of a phosphoryl group between substrates

Hydrolases • These enzymes catalyze cleavage reactions while using water • Peptidases, esterases, lipases, glycosidases, phosphatases are all examples of hydrolases named depending on the type of bond cleaved

Proteases • A class of hydrolytic enzymes is proteases • These enzymes catalyze proteolysis, the hydrolysis of a peptide bond within proteins • Proteolytic enzymes differ in their degree of substrate specificity

Exmaples • Trypsin, a digestive enzyme, is quite specific and catalyzes the splitting of peptide bonds only on the carboxyl side of lysine and arginine residues • Thrombin, an enzyme that participates in blood clotting, catalyzes the hydrolysis of Arg-Gly bonds in particular peptide sequences only

Lyases • These enzymes remove groups from their substrates resulting in the formation or removal of double bonds between C-C, C-O and C-N by a means other than hydrolysis

Types of lyases • Dehydrases: Removal of H 2 O from substrate to give double bond (example: enolase) • Decarboxylases: Replacement of a carboxyl group by a hydrogen • (example: pyruvate decarboxylase) • Synthases: Addition of a small molecule to a double bond (example: citrate synthase)

Isomerases • These enzymes catalyze intramolecular rearrangements

Ligases • Ligases join C-C, C-O, C-N, C-S and C-halogen bonds • The reaction is usually accompanied by the consumption of a high energy compound such as ATP and other nucleoside triphosphates • Example: pyruvate carboxylase, which catalyzes: Pyruvate + HCO 3 - + ATP Oxaloacetate + ADP + Pi

HOW DO SUBSTRATES FIT INTO THE ACTIVE SITE OF ENZYMES?

Lock-and-key model • The first is known as lock-and-key model where the substrate fits directly into the active site.

Induced fit model • The other model known as induced fit model states that enzymes are flexible and that the shapes of the active sites can be changed by the binding of substrate.

Activation energy • Conversion of a substrate to a product requires an energy called activation energy. • Enzymes accelerate reactions by lowering this energy. Binding of substrate to an enzyme makes it less stable

ENZYME REGULATION

Mechanisms of regulation • Non-specific inhibition (p. H and temperature) • Regulation of substrate and enzyme amount • Location (Compartmentalization and complexing of enzymes) • Expression of enzyme type (isoenzymes) • Regulation of enzymatic activity • • Inhibitors Allostery Reversible covalent modification Irreversible covalent modification

Effect of substrate Concentration • The rate increases with more substrate added to the reaction because more enzyme molecules are bound by the substrate. – If the substrate concentration doubles, the rate of the reactions doubles. • But: the increase in the rate begins to level off because the active sites are occupied (they become saturated). The rate of the reaction is determined by the efficiency of the enzyme, the p. H, and the temperature

Saturation

Effect of Enzyme Concentration • If the enzyme concentration doubles, the rate doubles; if the enzyme concentration • triples, the rate triples; and so on.

Regulation of enzyme amount • There are basically two mechanisms: – controlling rate of enzyme synthesis at the gene level – controlling rate of enzyme degradation by proteases

Temperature • Usually, the reaction rate increases • But, at high temperatures, the activity decreases because the protein part of the enzyme begins to denature • Enzymes have an optimal temperature – For humans, it is 37°C – For thermophilic bacteria, it can be 65°C

Hypothermia • A severe drop in body temperature creates the potentially fatal condition of hypothermia, which is accompanied by a slowdown in metabolic reactions. • This effect is used to advantage by cooling the body during cardiac surgery. • Upon gentle warming, enzymatic reaction rates return to normal because cooling does not denature proteins.

p. H • p. H can alter binding of substrate to enzyme by altering the protonation state of the substrate and/or altering the conformation of the enzyme • The effect of p. H is enzyme-dependent

Enzymes can be placed in small cellular compartments • For example, lysosomes contain many of the proteolytic enzymes responsible for protein degradation • Another example, synthesis of fatty acids are located in cytosol, whereas enzymes responsible for oxidation (break-sown) of fatty acids are located in the mitochondria

Enzymes can be made of multiple enzymes • The enzymes involved in a reaction sequence form a multienzyme complex – This allows the product of enzyme A to be passed directly to enzyme B • Example: Pyruvate dehydrogenase Pyruvate + Co. A + NAD+ Acetyl Co. A + CO 2 + NADH

Allostery • Allosteric regulation: binding of one molecule to one site changes binding of another molecule on a different site • Allosteric enzymes tend to be a multi-subunit • Allosteric enzymes contain an active site and a regulatory site • The binding of regulatory molecules causes conformational changes in the active site

Allosteric control Negative allosteric regulation Positive allosteric regulation

Enzyme inhibitors • The activity of many enzymes can be inhibited by the binding of specific small molecules. • Enzyme inhibition can be either reversible or irreversible. • Physiological inhibitors are reversible.

Irreversible inhibitors • An irreversible inhibitor tightly binds to the enzyme preventing the substrate from binding. • The inhibitor forms a bond that is not easily broken with a group in an active site. • Examples: – Heavy metals like mercury bind to –SH groups of cysteine – Sarin (nerve gas) binds to serine in the active site of acetylcholinesterase inhibiting it (no ore nerve impulses, paralysis)

Reversible inhibitors • Reversible inhibition is characterized by a rapid dissociation of the enzyme-inhibitor complex • Usually these inhibitors bind to enzymes by noncovalent forces

Types of reversible inhibitors Competitive Noncompetitive • The inhibitor competes with the substrate for the active site • the inhibitor binds at a site other than the catalytic site

Reversible covalent modification • A small group is added to the enzyme affecting its function. • Advantage: fast and transient regulation of enzyme activity The addition (phosphorylation) or removal (dephosphorylation) of a phosphate group by enzymes may activate or inactivate proteins like other enzymes

Irreversible covalent modification (proteolytic activation) • Many enzymes are synthesized as inactive precursors called zymogens or proenzymes • When a substrate is present, part of the enzyme is removed to make it an active enzyme • However, it is an irreversible process, so once the pro region is removed, it cannot go back

Examples of zymogens (digestive enzymes)

How does it work? • Three of the enzymes that digest proteins in the small intestine are produced in the pancreas as the zymogens trypsinogen, chymotrypsinogen, and proelastase. • These enzymes are inactive when they are synthesized so that they do not digest the pancreas. • Each zymogen has a polypeptide segment at one end that is not present in the active enzymes. • The extra segments are snipped off to produce trypsin, chymotrypsin, and elastase, the active enzymes, when the zymogens reach the small intestine, where protein digestion occurs.

Acute pancreatitis • One danger of traumatic injury to the pancreas or the duct that leads to the small intestine is premature activation of these zymogens, resulting in acute pancreatitis, a painful and potentially fatal condition in which the activated enzymes attack the pancreas.

Isoenzymes (isozymes) • These are enzymes that – Are produced by different genes – Can act on the same substrate(s) producing the same product(s) – Have different tissue distribution – Have different mechanisms of regulation – May have different catalytic activity

LDH • Lactate dehydrogenase (LDH) is a tetrameric enzyme composed of two protein subunits; the subunits are known as H (for heart) and M (for skeletal muscle) • These subunits combine in various combinations leading to 5 distinct isozymes (LDH 1 -5) with different combinations of the M and H subunits • The all H isozyme is characteristic of that from heart tissue, and the all M isozyme is typically found in skeletal muscle and liver

MODES OF REGULATION

Feedback inhibition • A common type of control occurs when an enzyme present early in a biochemical pathway is inhibited by a late product of pathway • This is known as feedback inhibition or negative feedback regulation

Positive feedback regulation • Enzymes can also be subject to positive feedback regulation where a product stimulates the activity of an enzyme

Feed-forward regulation • A third mechanism is feed-forward regulation where a substrate produced early in a pathway activates an enzyme downstream of the same pathway

A committed step • A committed step is an irreversible reaction that, once occurs, leads to the formation of a final substrate with no point of return • For example, the committed step for making product E is (B → C), not (A → B)

Rate-limiting reactions • Some reactions are called rate-limiting since they limit rate (speed) of reactions. • Why do they limit reactions? – They need lots of energy. – They are highly regulated.

ENZYMES IN DISEASE DIAGNOSIS

Concept • The measurement of the serum levels of numerous enzymes has been shown to be of diagnostic significance • This is because the presence of these enzymes in the serum indicates that tissue or cellular damage has occurred resulting in the release of intracellular components into the blood

Information from enzymes measurements in serum Presence of disease Organs involved Etiology /nature of disease: differential diagnosis Extent of disease-more damaged cells-more leaked enzymes in blood • Time course of disease • •

Measurement of enzyme activity • Enzyme activity is expressed in International unit (IU) • It corresponds to the amount of enzymes that catalyzes the conversion of one micromole ( mol) of substrate to product per minute

Enzymes used in the clinic ENZYME PRESENT IN Aspartate Amino transferase (AST) Serum glutamate-oxaloacetate transaminase (SGOT) Heart and Liver Alanine Amino transferase (ALT) Serum glutamate-pyruvate transaminase (SGPT) Heart and Liver Alkaline Phosphatase (ALP) Bone, intestine and other tissues Acid Phosphatase (ACP) Prostate glutamyl Transferase ( GT) Liver Creatine kinase (CK) Muscle Including cardiac muscle Lactate Dehydrogenase (LDH) Heart, liver, muscle, RBC Amylase Pancreas

AST and ALT • The typical liver enzymes measured are AST and ALT • ALT is particularly diagnostic of liver involvement as this enzyme is found predominantly in hepatocytes. • When assaying for both ALT and AST the ratio of the level of these two enzymes can also be diagnostic. • Normally in liver disease or damage that is not of viral origin the ratio of ALT/AST is less than 1. • However, with viral hepatitis the ALT/AST ratio will be greater than 1.

ALKALINE PHOSPHATASE (ALP) • Is a group of enzymes that have maximal activity at a high p. H 9. 0 -10. 5 • High levels are seen is liver and bone, and useful to assess hepatobiliary and bone diseases • In liver, high levels of ALP is indicative of extrahepatic obstruction • In bones, the enzyme is increased in bone diseases like rickets, osteomalacia, neoplastic diseases with bone metastates and healing fractures

ACID PHOSPHATASE (ACP) • Is a group of enzymes that have maximal activity at p. H 5. 0 -6. 0 • It is present in prostate gland. • The main source of ACP is prostate gland so can be used as a marker for prostate disease.

AMYLASE • Is the digestive enzymes from the pancreas and salivary glands to digest complex carbohydrates. • Elevated in acute pancreatitis. • It is used as a marker to detect acute pancreatitis and appendicitis.

-Glutamyltransferase ( -GT) • Amino acid + Glutathione -glutamyl amino acid + Cysteinylglycine • Found mainly in biliary ducts of the liver, kidney and pancreas. • Enzyme activity is induced by a number of drugs and in particular alcohol. • -GT increased in liver diseases especially in obstructive jaundice. • -GT levels are used as a marker of alcohol induced liver disease and in liver cirrhosis.

Myocardial infarction

CPK • CPK is found primarily in heart and skeletal muscle as well as the brain. Therefore, measurement of serum CPK levels is a good diagnostic for injury to these tissues • Like LDH, there are tissue-specific isozymes of CPK: – CPK 3 (CPK-MM) is the predominant isozyme in muscle – CPK 2 (CPK-MB) accounts for about 35% of the CPK activity in cardiac muscle, but less than 5% in skeletal muscle. – CPK 1 (CPK-BB) is the characteristic isozyme in brain and is in significant amounts in smooth muscle

CPK and myocardial infarction • Since most of the released CPK after a myocardial infarction is CPK-MB, an increased ratio of CPK-MB to total CPK may help in diagnosis of an acute infarction, but an increase of total CPK in itself may not

LDH • Lactate dehydrogenase exists in 5 types differentially and specifically distributed in tissues • The 5 isoforms of LDH: LDH 1, 2, 3, 4, and 5 • LDH is especially diagnostic for myocardial infarction (heart attacks) among other diseases like acute hepatitis (LDH 5 > LDH 4).

LACTATE DEHYDROGENASE IN MI

LDH 1 vs. LDH 2 • A characteristic change following a heart attack is an elevated level of LDH 1 above LDH 2. • A comparison of serum levels of LDH-1/LDH-2 ratio is diagnostic • Normally, this ratio is less than 1 • Following an acute myocardial infarct, the LDH ratio will be more than 1.