Cellular Biology & Homeostasis - Enzymes Part 1

Learning Objectives

• Define enzyme and the key-lock mechanism

• List some industrial applications of enzymes and give examples

• Describe how enzymes work, including activation energy and the transition state

• Understand enzyme nomenclature and give examples

• Define apoenzyme, holoenzyme, and cofactors

• List and explain the properties of enzymes

Recap of Digestive Enzymes

• Salivary glands:

  • α-amylase (Ptyalin): Amylose (polysaccharide) $→$ disaccharides

  • Lingual lipase: Lipids (TAG, cholesterol) $→$ DAG, MAG, FFA, glycerol

• Stomach:

  • Pepsin (protease): Proteins $→$ peptides

  • Gastric lipase: Lipids $→$ DAG, MAG, FFA, glycerol

• Pancreas:

  • Pancreatic amylase: Polysaccharides $→$ disaccharides

  • Trypsin (protease): Proteins $→$ peptides

  • Chymotrypsin (protease): Proteins $→$ peptides

  • Acid lipases: Lipids $→$ DAG, MAG, FFA, glycerol

• Small intestine brush border:

  • Peptidases: Polypeptides $→$ amino acids

  • Nucleotidases, nucleases: DNA, RNA $→$ nucleotides, ribose

  • Lactase: Disaccharides $→$ monosaccharides

  • Maltase: Disaccharides $→$ monosaccharides

  • Sucrase: Disaccharides $→$ monosaccharides

Enzymes as Mediators

• Enzymes act as mediators for virtually all chemical reactions in biological systems.

• They play fundamental roles in:

  • Metabolic events

  • Signal transduction

  • Cell regulation

General Concepts

• Enzymes are proteins that act as biological catalysts by accelerating chemical reactions.

• Substrates are the molecules upon which enzymes act, converting them into products.

• Enzymes exhibit a high degree of specificity for their substrates.

• They accelerate chemical reactions tremendously without being changed or used up during the process (reversible binding).

• A small amount of enzyme can influence a large amount of reactive substrate.

Enzyme Specificity

• The binding is very specific; small changes in the shape of the ligand/substrate (key) can cause a major change in protein (lock) behavior.

• Complementary shape is crucial for recognition and plays a major role in information transfer.

Allostery

• Allostery: The ability of a protein to change shape, resulting in a change in binding affinity at a different binding site. Shape influences binding, and in turn, binding can influence shape.

• Allosteric enzymes have the active site as well as an additional site (allosteric site).

• From the Greek 'allo', which means 'other'.

Importance of Enzyme Catalysis

• Almost all metabolic processes in the cell need enzyme catalysis to occur at rates fast enough to sustain life.

• Catalyzed reaction: $E + S \rightleftharpoons ES \rightarrow EP \rightarrow E + P$

• Spontaneous reaction: $S \rightarrow P$

• Binding sites are usually very specific for a particular ligand/substrate, and the binding is reversible.

Industrial Applications of Enzymes

• Biofuel production (biodiesel, alcohol from sugar cane)

• Agricultural (animal feed additives, fertilizers)

• Fermentations: transformation of raw materials such as sugar, starch, etc., in industrial mixtures such as liquors, brewing

• Biotransformations: transformation of defined precursors to a desired target product

  • Environmentally friendly processes to treat waste

• Pharmaceutical industry: synthesis & modification of antibiotics & medicines

• Diagnosis of disease: Increased or decreased concentrations of enzyme activity in the target system (liver, kidney, muscle)

• Treatment of disease: i.e., use of streptokinase to dissolve blood clots, exocrine pancreatic insufficiency

Enzymes in Industrial Applications - By Sector

• Pharmaceuticals

  • Enzymes: Nitrile hydratase, transaminase, monoamine oxidase, lipase, penicillin acylase

  • Applications: Synthesis of intermediates for the production of active pharmaceutical ingredients

• Food processing

  • Enzymes: Trypsin, amylase, glucose isomerase, papain, pectinase

  • Applications: Conversion of starch to glucose, production of prebiotics, debittering of fruit juice

• Detergent

  • Enzymes: Protease, lipase, amylase, cellulase

  • Applications: Stain removal, removal of fats and oils, color retention

• Biofuels

  • Enzymes: Lipase, cellulase, xylanase

  • Applications: Production of fatty acid methyl esters, decomposition of lignocellulotic material for bioethanol production

• Paper and pulp

  • Enzymes: Lipase, cellulase, xylanase

  • Applications: Removal of lignin for improved bleaching, improvement in fiber properties

Some Industrial Enzymes and Their Uses

• Protease: Degradation of proteins in detergents

• Cellulase: Degradation of cellulose in detergents

• Lipase: Degradation of lipids in detergents

• Amylase: Conversion of starch to glucose in starch processing

• Glucose Isomerase: Production of High Glucose Syrup in starch processing

• Phytase: Improve nutrient availability in animal feed

• Xylanase: Removal of lignin ‘bio bleaching’ in paper and pulp

• Amylase: Removal of fruit starch haze in fruit/vegetable processing

• Hydrolase: Breakers for biopolymer gels in Petrol and Gas

• Chymosin: Clotting in cheese manufacture in dairy

• Pectinase: Increased yields in wine production

How Enzymes Work

• Chemical reactions have an energy barrier separating the reactants and the products.

• Energy is needed to get them started = Activation energy

• Enzymes greatly reduce the activation energy barriers that block chemical reactions.

Enzyme Pathways

• Enzymes direct substrate molecules through a specific reaction pathway.

• Allowing a reaction to proceed rapidly by providing an alternate reaction pathway in the cell which has a lower activation energy.

• Enzymes show a high selectivity and usually catalyze only one specific reaction, or a set of closely related reactions; directing a particular reaction pathway.

The Transition State

• The active site acts as a molecular template that binds the substrate and initiates its conversion to the transition state.

• The transition state is the form the substrate must take before it becomes product.

• It is the highest energy point of the reaction.

• What is:

  • $\triangle G^{\ddagger}uncat$? (Gibbs free energy of activation for the uncatalyzed reaction)

  • $\triangle GM$?

  • $\triangle G^{\ddagger}$? (Gibbs free energy of activation for the catalyzed reaction)

Accelerating Reactions

• Stabilizing the transition state (T*) an enzyme can greatly increase the concentration of the reactive intermediate that can be converted to product accelerating the reaction.

Nomenclature

• Recommended name: short name, most used, has the suffix ‘-ase’ attached to:

  • The substrate of the reaction: i.e., Glucokinase (found mostly in liver and pancreas, phosphorylation of glucose)

  • The description of the reaction performed: i.e., Lactate dehydrogenase

• Systematic name: more complete, complex; is used when an enzyme must be identified without ambiguity.

• The suffix -ase is attached to a more complete description of the chemical reaction catalyzed, including the names of all substrates: LDH (lactate dehydrogenase): Lactate, NAD+ oxidoreductase

• The systematic names are unambiguous and informative, but often too big for general use

Nomenclature - Trivial Names

• Some enzymes retain their original, trivial names, which give no hint of the associated enzymatic reaction.

• Examples:

  • Catalase (Substrate: Hydrogen peroxide ($H<em>2O</em>2$), Products: Oxygen gas + water ($O<em>2 + H</em>2O$))

  • Diastase (Substrate: Starch, Products: Maltose)

  • Pectinase (Substrate: Pectin in plant cell walls, Products: Simple sugars (releases juices from cells))

  • Pepsin (Substrate: Protein, Products: Short polypeptides)

  • Rennin (Substrate: Soluble casein (milk protein), Products: Insoluble casein (curdled milk))

The Major Classes of Enzymes

• Oxidoreductases: Catalyze reactions in which one molecule is oxidized while the other is reduced, transfer of electrons ($e^-$) and hydrogens $H^+$

  • oxidases, reductases, dehydrogenases, peroxidases

• Transferases: Transfer carbon, nitrogen, or phosphate groups

  • methyltransferases, aminotransferases, kinases, phosphorylases

• Hydrolases: Enzymes that catalyze a hydrolytic cleavage reaction (use water to break a chemical bond)

  • most digestive enzymes are hydrolases

  • nucleases, proteases, phosphatases, amylase, lipase

• Lyases: Catalyze the cleavage of C-C, C-S, and C-N bonds (catalyzes the breaking of various chemical bonds by means other than hydrolysis and oxidation, often forming a new double bond or a new ring structure)

  • decarboxylases, aldolases, synthases, polymerases

• Isomerases: Catalyze the rearrangement of bonds within a single molecule, transfer of groups within molecules to yield isomeric forms

  • mutases, racemases

• Ligases: Join two molecules in an energy-dependent process

  • Catalyze formation of bonds between carbon and O, S, and N coupled to hydrolysis of high energy phosphates

Classes of Enzymes and Nomenclature

• Synthetase (ligase class): requires ATP

• Synthase (lyase class): no ATP required

• Phosphatase (hydrolase class): remove phosphates

• Phosphorylase (transferase class): transfer (add) inorganic phosphates

Potentially Confusing Enzyme Nomenclature:

• Oxidoreductases

  • Dehydrogenase: catalyze oxidation/reduction reactions (i.e., transferring hydrogen to $NAD^+/NADPH^+$

  • Oxidase: $O_2$ is the acceptor of electrons or hydrogen, and oxygen atoms are not incorporated into substrate

  • Oxygenase: catalyze the incorporation of molecular $O_2$ to a substrate.

Enzymes Mostly Discussed in Lectures

• Digestive enzymes – Proteases, Lipase, and Amylase (hydrolase class):

  • break down proteins, lipids, and carbs by hydrolyzing bonds

• Kinases (transferase class):

  • Catalyze the addition of phosphate groups (from ATP) to molecules

  • (protein kinases are very common in physiology)

• ATPases:

  • Hydrolyze ATP (Na/K- ATPase pump)

• Phosphatase:

  • catalyze the hydrolytic removal of a phosphate group from a molecule

Properties and Characteristics of Enzymes

• Active sites: enzymes contain a special pocket called the ‘active site’ which has a high specificity

  • Contains amino acid side chains that participate in substrate binding and catalysis

  • “Reusable”

  • Sensitive to pH changes

  • Denatured by high heat

  • Inhibited by poison/toxin/heavy metals

• Catalytic Efficiency: reactions catalyzed by enzymes are $10^3-10^8$ times faster than uncatalyzed reactions.

• Specificity: enzymes interact with one or very few substrates and catalyze only one type of chemical reaction.

• Presence of Cofactor and coenzymes

  • Coenzymes and Cosubstrates are often the metabolically active form of the vitamins.

Enzyme Location and Regulation

• Location in the cell: Many enzymes are in specific organelles in the cell (compartmentalization) and in specific cells.

  • some reactions are isolated from others (avoiding competition for the substrate or enabling more favorable conditions, like pH)

    • Glycolysis

    • PP pathway

    • Fatty acid synthesis

  • Recall: protein sorting importance to maintain this compartmentalization.

• Regulation: enzyme activity can be regulated (it can be increased or decreased) so that the rate of product (biochemical reactions) responds to cellular needs

  • recall: insulin and glucagon regulating several enzymatic activities on metabolic map pathways

Serum Biochemistry - Enzyme Diagnosis

• Cellular enzymes are released in blood plasma:

  • Actively secreted (i.e., liver secretes zymogens involved in blood coagulation)

  • Released from normal cellular turnover (usually have no physiologic use in the blood plasma)

• In healthy individuals:

  • The rate of release from cellular turnover is balanced by an equal rate of removal from blood plasma

• Alteration of plasma/serum enzyme levels may indicate tissue damage (disease states)

  • Serum is the liquid that remains after the clotting of blood

  • Plasma is the liquid that remains when anticoagulant is added to prevent clotting

Serum Biochemistry - Enzyme Diagnosis - FYI

• Liver Enzymes:

  • ALT (alanine aminotransferase) (typically found when the cells of the liver are stressed or damaged)

  • ALP (alkaline phosphatase) (increased when bile flow in the liver is reduced)

• Pancreatic Enzymes:

  • Amylase

  • Lipase

  • PLI (pancreatic lipase immunoreactivity)

• Muscle Enzymes:

  • CK (creatine kinase) enzyme most frequently measured to assess injury (trauma, inflammation).

  • AST (aspartate aminotransferase) and ALT (alanine aminotransferase) also used to assess liver function, lesser importance during muscle injury.

• Testing for specific enzymes provides information about the organs and tissues in the body as well as the metabolic state of the animal.