RPI-BIOL-2120 Lecture 3: Enzymes & Biochemical Pathways (PDF)

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Rensselaer Polytechnic Institute

Dr. Michael T. Klein

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enzymes biochemistry cellular pathways biology

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This document is a lecture from a university-level cell biology course, focusing on enzymes and biochemical pathways. It provides a comprehensive overview of metabolic and energy-related processes in cells.

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BIOL-2120 INTRODUCTION TO CELL & MOLECULAR BIOLOGY Dr. Michael T. Klein ([email protected]) LECTURE 3 ENZYMES & BIOCHEMICAL PATHWAYS Reference text: Essential Cell Biology, 5th ed., Alberts et al. 2019. Chapter 3...

BIOL-2120 INTRODUCTION TO CELL & MOLECULAR BIOLOGY Dr. Michael T. Klein ([email protected]) LECTURE 3 ENZYMES & BIOCHEMICAL PATHWAYS Reference text: Essential Cell Biology, 5th ed., Alberts et al. 2019. Chapter 3 Images provided by M. T. Klein or W. W. Norton & Company unless stated otherwise MTK, 2025-01-06 (1) METABOLISM AND THE CELLULAR ENVIRONMENT  Metabolism refers to all of the chemical reactions that occur within an organism and its cells, and these reactions can be divided into two types of pathways: Catabolic pathways are those that break down molecules to release energy and produce intermediate metabolites (smaller molecules that can be used to build up larger ones) Anabolic pathways are those that consume energy to build smaller molecules into larger ones and establish order (i.e., decrease entropy) within the living organism  Enzymes are biological catalysts that facilitate metabolic reactions, but they add no energy to the reactions; they only increase the speed at which reactions may take place MTK, 2025-01-06 (2) HOW CELLS USE ENERGY  To understand metabolism, you must track the energy flow within an organism and its cells  Biological systems obey the laws of thermodynamics: 1. Conservation of energy: energy can neither be created nor destroyed ꟷ In order for work to occur, energy must be put into the system 2. Entropy (disorder) tends to increase over time: energy flows down gradient, i.e., from high to low ꟷ Energy transfer or transformation increases the total entropy of the universe ꟷ Energy is always required to create organization (i.e., to decrease entropy) ꟷ E.g., organized metabolic pathways, cytoskeleton structure, organelles, DNA sequences, active transport 3. Absolute zero is unattainable: in other words, heat energy is always present Heat is a form of kinetic energy MTK, 2025-01-06 (3) HOW CELLS USE ENERGY TYPES OF ENERGY  Energy is the capacity to cause change (to do work)  Energy exists in various forms and falls into 1 of 2 categories: Kinetic energy is energy associated with motion ꟷ Heat is kinetic energy associated with the random movement and vibrations of matter; a system becomes hotter as the rate of molecular motion increases Potential energy is energy that matter possesses because of its structure or location ꟷ Chemical energy is the potential energy stored in a molecule’s bonds and can be released in a chemical reaction ꟷ Molecules or ions concentrated on one side of a membrane separating different compartments of a cell or the inside of a cell from the outside » Why would concentration matter? » How can this energy be harnessed to do work? What kind of work? MTK, 2025-01-06 (4) HOW CELLS USE ENERGY HEAT  The second law of thermodynamics dictates that energy is consumed to counter entropy (i.e., to establish and maintain order)  But the first law dictates that the energy consumed by a cell cannot just disappear Consider the growth of a cell and the building of complex molecules, but keep in mind that a cell continues to consume energy just to maintain its structures If energy is consumed by the cell, it must leave the cell in another form  So, for biological order to exist, heat must be released from cells as waste energy As one part of the universe gets more ordered or stays ordered (inside of a cell) another part of the universe must become less ordered (outside of the cell) MTK, 2025-01-06 (5) HOW CELLS USE ENERGY CONVERSION OF ENERGY  Not all forms of energy are useful to a cell for all purposes, cells can convert energy from one form to another; e.g.: The energy stored in the bonds of a sugar molecule cannot be directly harnessed to pump ions across a membrane But the bonds of a sugar molecule can be broken and new bonds formed to make the molecule ATP, which can be used by an ion transporter to pump ions In this process, small molecule waste products are produced (e.g., CO2, H2O) and carry away waste energy as heat MTK, 2025-01-06 (6) HOW CELLS USE ENERGY CONVERSION OF ENERGY  Cells that can carry out photosynthesis can use the energy of sunlight to drive biochemical reactions that transform electromagnetic energy (photons) into chemical energy (covalent bonds of sugars)  To accomplish this, many intermediate reactions are required where energy must be ferried from one step to the next MTK, 2025-01-06 (7) HOW CELLS USE ENERGY REDOX REACTIONS  Oxidation and reduction reactions (redox reactions) involve electron transfers; these reactions are used to ferry energy from one reaction to the next MTK, 2025-01-06 (8) HOW CELLS USE ENERGY REDOX REACTIONS  A molecule is oxidized when it loses an electron or its bonds become more polar  A molecule is reduced when it gains electrons (often times accompanied by a hydrogen atom) or its bonds become less polar – this requires energy input, thus, the energy stored in a reduced molecule can be used to do work  E.g., methane is a nonpolar molecule but can be oxidized to CO2 which has more polar bonds – burning of methane can be used to do work, but CO2 is a waste product MTK, 2025-01-06 (9) HOW CELLS USE ENERGY ENERGY FLOW THROUGH THE BIOSPHERE  Because photosynthesizers can take in sunlight and convert this energy into useful organic molecules, they support nearly the entire biosphere and its energy needs  But remember the first law of thermodynamics: If photosynthesizers are taking in energy from the sun, here does all of this energy end up? Can this energy be stored on Earth?  And consider: Do all photosynthesizers feed the biosphere? Are photosynthesizers the only types of organisms that can feed the biosphere? MTK, 2025-01-06 (10) FREE ENERGY AND CATALYSIS  In complex reactions, the equilibrium constant (K) includes the concentrations of all reactants and products K will tell you the concentrations of reactants and products when equilibrium is reached  K and the change of free energy (ΔG) are in a fixed relationship – at equilibrium the free energy (G) of the system is at its lowest, but not zero Why is G not 0 at equilibrium?  For sequential reactions (e.g., metabolic pathways), the changes in free energy are additive  Noncovalent interactions allow enzymes to bind specific molecules to catalyze reactions  Enzyme-catalyzed reactions depend on rapid molecular collisions MTK, 2025-01-06 (11) FREE ENERGY AND CATALYSIS GIBB’S FREE ENERGY (G)  Reactions that occur spontaneously are called exergonic reactions and require no energy input  Reactions that do not occur spontaneously require an input of energy and are called endergonic reactions Endergonic reactions will not go forward on their own, but with the addition of energy (usually the breaking of covalent bonds), the total reaction becomes exergonic and will occur spontaneously  The change of Gibb’s free energy is written as ΔG; this value tells use how much energy must be put into the system for the reaction to go forward – it tells us whether or not the reaction occurs spontaneously What does it mean when ΔG is positive? Negative? MTK, 2025-01-06 (12) FREE ENERGY AND CATALYSIS GIBB’S FREE ENERGY (G)  Terminology: G refers to Gibb’s Free Energy G refers to change in Gibb’s Free Energy Enthalpy (∆H) is the total change in energy Entropy (∆S) is the change in total disorder  The change in free energy (∆G) during a process is related to the changes in enthalpy (∆H) and entropy (∆S) and temperature in degrees Kelvin (T): ∆G = ∆H – T∆S What is Kelvin? Why use Kelvin here?  Only processes with a negative ∆G are spontaneous (exergonic) Meaning that in chemical reactions the products of the reaction are in a lower energy state than the reactants MTK, 2025-01-06 (13) FREE ENERGY AND CATALYSIS GIBB’S FREE ENERGY (G)  To review: The concept of free energy can be applied to the chemistry of life’s processes An exergonic reaction proceeds with a net release of free energy (-∆G) and is spontaneous An endergonic reaction absorbs free energy from its surroundings (+∆G) and is nonspontaneous MTK, 2025-01-06 (14) FREE ENERGY AND CATALYSIS GIBB’S FREE ENERGY (G)  Spontaneous processes can be harnessed to perform work – they can be used to drive other processes forward E.g., hydrolyzing ATP (a reaction where ∆G is negative) is used to drive the synthesis of amino acids (a reaction where ∆G is positive) ꟷ In a closed (ideal) system the ∆G = 0 for the total reaction ꟷ The first law of thermodynamics is satisfied in this way  Remember, just because a reaction is spontaneous (-∆G) it does not mean the reaction will be rapid Think of a car rolling down a gentle hill vs. a steep hill, the car will spontaneously roll without any energy input but the car on the gentle hill will roll much more slowly than the other car MTK, 2025-01-06 (15) FREE ENERGY AND CATALYSIS COUPLED REACTIONS  Spontaneous processes proceed towards equilibrium Equilibrium is a state of maximum stability  Reactions in a closed system eventually reach equilibrium and then no longer do work A dead cell is at equilibrium, it can do no work, it cannot maintain order, thus it is dead  Metabolism is never at equilibrium (some reactions will be at equilibrium, but all reactions in total are not)  Cells are open systems experiencing a constant flow of materials into and out of the cell  A catabolic pathway of a cell releases free energy in a series of reactions, one reaction driving the next reaction MTK, 2025-01-06 (16) FREE ENERGY AND CATALYSIS ENERGY COUPLING  A cell does three main kinds of work going on within cells: Chemical – making and breaking bonds Transport – movement of material around a cell into/out of the cell or cellular compartments Mechanical – exerting force  To do work, cells manage resources by energy coupling – the use of an exergonic process to drive an endergonic one  Most energy coupling in cells is mediated by the molecule ATP ATP powers cellular work by coupling exergonic ATP hydrolysis to endergonic reactions MTK, 2025-01-06 (17) FREE ENERGY AND CATALYSIS ENERGY COUPLING  ATP is composed of ribose (a sugar), adenine (a nitrogenous base), and three phosphate groups  Hydrolysis of the terminal phosphate group is spontaneous, releasing 7.3 kcal/mol I.e., the ∆G is -7.3 kcal/mol  This energy can be used to drive an endergonic reaction forward – the coupling of ATP hydrolysis and an endergonic reaction results in an overall exergonic reaction MTK, 2025-01-06 (18) FREE ENERGY AND CATALYSIS ENERGY COUPLING MTK, 2025-01-06 (19) FREE ENERGY AND CATALYSIS ACTIVATION ENERGY  Spontaneous reactions will always proceed, but even with a very negative ΔG, this does not indicate the reaction will be rapid  This is because a reaction must overcome its activation energy (EA) barrier – some amount of energy must be put in so that a much larger amount of energy is released  A catalyst is an agent that reduces the activation energy required for the reaction MTK, 2025-01-06 (20) ENZYMES  Enzymes are biological catalysis built out of proteins Some enzymes are very simple, even just being composed of a single polypeptide, but many enzymes are built from multiple polypeptide subunits Most enzymes require additional cofactors like ions and other organic compounds (coenzymes) to function  Ribozymes are biological catalysis built out of RNA molecules MTK, 2025-01-06 (21) ENZYMES  The molecule(s) that an enzyme acts upon is called the enzyme’s substrate  The enzyme binds to its substrate, forming an enzyme- substrate complex  The active site is the region on the enzyme where the substrate binds  Induced fit of a substrate brings chemical groups of the active site into positions that enhance their ability to catalyze the reaction The concept of “Lock-and-Key” to describe the enzyme- substrate complex is antiquated but still appears in many textbooks MTK, 2025-01-06 (22) ENZYMES LOWERING THE EA BARRIER  The active site can lower an EA barrier by: Orienting substrates correctly Straining substrate bonds Providing a favorable microenvironment Covalently bonding to the substrate ꟷ This covalent bond is later broken so the enzyme will remain functional for subsequent reactions MTK, 2025-01-06 (23) ENZYMES ACTIVITY AND REGULATION  An enzyme’s activity can be affected its environment  Optimal conditions favor the most active shape of the enzyme  Each enzyme has an optimal temperature The optimal temp. is the temp. in which the enzyme has evolved to operate in; e.g., most human enzymes function best at 37°C  The same is true for optimal pH and salinity MTK, 2025-01-06 (24) ENZYMES ACTIVITY AND REGULATION  Cofactors are non-protein enzyme helpers Cofactors help substrates bind to the active site or bind to another part of the enzyme to alter its conformation to improve enzyme-substrate associations  Cofactors may be inorganic, such as a metals in ionic form, like magnesium (Mg2+)  Cofactors can also be organic and are called coenzymes Most vitamins are coenzymes or are used by cells to synthesize coenzymes  The enzyme on its own, without its cofactors is called an apoenzyme and is inactive; when the cofactors are present it is called a holoenzyme MTK, 2025-01-06 (25) ENZYMES ENZYME INHIBITORS  Competitive inhibitors are molecules that bind to the active site of an enzyme, competing with the substrate If substrate concentration increases, the substrate can outcompete the inhibitor and the enzyme's maximal activity will be restored MTK, 2025-01-06 (26) ENZYMES ENZYME INHIBITORS  Noncompetitive inhibitors come in two types: Irreversible inhibitors covalently bind to the enzyme’s active site, permanently blocking substrate from entering it; enzyme activity cannot be restored no matter how much substrate is present MTK, 2025-01-06 (27) ENZYMES ENZYME INHIBITORS  Noncompetitive inhibitors come in two types: Allosteric inhibitors bind to a region other than the active site (an allosteric site); they do not block association of substrate with the active site; they cause the enzyme to change shape to a less than optimal conformation MTK, 2025-01-06 (28) ENZYME ENZYME ALLOSTERISM  Compounds that bind to allosteric sites on enzymes may either be allosteric inhibitors (negative modulators) or allosteric activators (positive modulators)  Allosteric activators operate in a similar fashion as allosteric inhibitors: They bind to an allosteric site, causing the enzyme to change shape However, the shape the enzyme assumes after binding an activator is more favorable for catalysis  Enzyme allosterism is critical for metabolic regulation MTK, 2025-01-06 (29) ENZYMES ENZYME ALLOSTERISM  Many allosterically regulated enzymes have multiple subunits: An activator stabilizes the active form of the enzyme An inhibitor stabilizes the inactive form of the enzyme  Cooperativity can amplify enzyme activity – once a substrate binds to one subunit, the other subunits can bind substrate more readily Cooperativity Inactive Active MTK, 2025-01-06 (30) ENZYME FEEDBACK  In feedback inhibition, the end product of a metabolic pathway shuts down the pathway  Feedback inhibition prevents a cell from synthesizing more product than needed, preventing the waste of chemical resources  What would positive feedback look like? MTK, 2025-01-06 (31) ENZYMES COMPARTMENTALIZATION  Structures within the cell help bring order to metabolic pathways  Some enzymes act as structural components of membranes  In eukaryotic cells, some enzymes reside in specific organelles; e.g.: Enzymes for cellular respiration are located only in mitochondria Digestive enzymes are found in lysosomes MTK, 2025-01-06 (32) EXTRA CREDIT DISCUSSION: ACTIVATION ENERGY USE BLACKBOARD DISCUSSION BOARD TO POST ANSWERS  The term activation energy may sound nebulous at first – so I want you to think about what it actually means: Give a real-world (macroscopic) physical example of a process overcoming an energy barrier to ultimately output more energy that what was put in. Give a specific example where molecules or atoms within a cell physically overcome some barrier to their reaction going forward. The reaction must be an exergonic one. Remember to site your sources. ꟷ Hint: think about what a catalyst (enzyme) physically does during a reaction. MTK, 2025-01-06 (33) QUICK REFERENCE FREE ENERGY AND BIOLOGICAL MTK, 2025-01-06 REACTIONS (34)

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