Microbiology 7.1 - 7.2 (p. 168-177)

The Metabolic Role of ATP

*ATP is the primary energy currency of the cell*. ATP utilization and replenishment is an ongoing cycle. In many instances, the *energy released during ATP hydrolysis powers biosynthesis by activating individual subunits before they are enzymatically linked together*. ATP is also used to *prepare molecules for catabolism*, such as when a 6-carbon sugar is phosphorylated during the early stages of glycolysis: ATP --> ADP Glucose --> Glucose-6-phosphate When ATP is utilized, by the removal of the terminal phosphate to release energy plus ADP, ATP then needs to be re-created. The reversal of this process-- that is, adding the terminal phosphate to ADP, will replenish ATP, but it requires an input of energy: ATP <--> ADP + P1 + Energy In heterotrophs, the energy infusion that regenerates a high-energy phosphate comes from certain steps of catabolic pathways, in which nutrients such as carbohydrates are degraded and yield energy. ATP is formed when substrates or electron carriers provide a high-energy phosphate that becomes bonded to ADP.

Denaturation

*Denaturation* is a process by which the *weak bonds that collectively maintain the native shape of the apoenzyme are broken*. This disruption causes *extreme distortion of the enzyme's shape* and prevents the substrate from attaching to the active site. Such nonfunctional enzymes block metabolic reactions and thereby can lead to cell death. Low or high pH or certain chemicals (heavy metals, alcohol) are also denaturing agents.

Enzymes are classified into 6 classes based upon its biochemical action...

1. *Oxidoreductases* transfer electrons from one substrate to another, and *dehydrogenases* transfer a hydrogen from one compound to another. 2. *Transferases* transfer functional groups from one substrate to another. 3. *Hydrolases* cleave bonds on molecules with the addition of water. 4. *Lyases* add groups to or remove groups from double-bonded substrates. 5. *Isomerases* change a substrate into its isomeric form. 6. *Ligases* catalyze the formation of bonds with the input of ATP and the removal of water.

Assess Your Progress (1)

1. Describe the relationship among metabolism, catabolism, and anabolism. Metabolism pertains to all chemical reactions within a cell. Catabolism is the process of breaking down bonds from larger molecules into smaller molecules, which in turn releases energy. Anabolism is a bond-making process that forms larger macromolecules from smaller ones, and usually requires the input of energy. 2. Fully define the structure and function of enzymes. Enzymes have very specific active sites that are made to fit with specific substrates like a "lock-and-key." Enzymes are much bigger than the substrates for which they provide a platform for chemical reaction to take place. The function of enzymes is to speed up chemical reactions without getting used up in the process. 3. Differentiate between constitutive and regulated enzymes. Constitutive enzymes are always present and in relatively constant amounts, regardless of the amount of substrate. Regulated enzyme production can be either turned on (induced) or turned off (repressed) in response to changes in concentration of the substrate. 4. Diagram some different patterns of metabolism. There is competitive inhibition, where a competitive inhibitor (mimic) will compete with the normal substrate for the active site of the enzyme. Noncompetitive inhibition occurs on enzymes that possess both an active site and a regulatory site. If a regulator molecule binds to the regulatory site, the enzyme is changed, thus blocking the normal active site and slowing down the reaction. 5. Describe how enzymes are controlled. In enzyme repression, excess product will eventually bind to DNA, which will shut down the further production of that enzyme. Enzyme induction, however, depends on the substrate present. Whatever the substrate is will induce the synthesis of the corresponding enzyme.

Assess Your Progress (2)

6. Name the chemical in which energy is stored in cells. Adenosine Triphosphate, or, ATP. 7. Create a general diagram of a redox reaction. - Reducing agent gives up electrons. - Oxidizing agent accepts electrons. - The energy now present in the electron acceptor can be captured to phosphorylate to ADP or to some other compound. 8. Identify electron carriers used by cells. Electron carriers, or coenzyme carriers, used by cells are nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD).

How Do Enzymes Work?

A certain amount of energy is required to initiate the building or breaking of bonds in every such reaction. The rate of these reactions could be sped up by heat or other means, but cells use enzymes which *promotes reactions by serving as a physical site upon which the reactant molecules (called substrates) can be positioned for various interactions*. The enzyme is much larger in size than its substrate, and it presents a unique active site that fits only that particular substrate. Enzymes do not get used up in a reaction and the speed of enzymes differs in range.

The Molecular Structure of ATP (fig. 7.10)

ATP is a three-part molecule consisting of a nitrogen base (*adenine*) linked in a 5-carbon sugar (*ribose*), with a chain of *three phosphate groups* bonded to the ribose. The high energy of ATP originates in the *orientation of the phosphate groups*, which are relatively *bulky and carry negative charges*. The proximity of these repelling electrostatic charges imposes a strain that is most acute on the bonds between the last two phosphate groups. The *strain on the phosphate bonds accounts for the energetic quality of ATP because removal of the terminal phosphates releases free energy*. Breaking the bonds between two successive phosphates of ATP yields adenosine diphosphate (*ADP*), which is then converted to adenosine monophosphate (*AMP*). AMP derivatives help form the backbone of RNA and are also a major component of certain coenzymes (NAD, FAD, and coenzyme A).

Each enzyme is also assigned a common name that indicates the specific reaction it catalyzes...

An enzyme that digests a carbohydrate substrate is a *carbohydrase*; a specific carbohydrase, amylase, acts on starch. The enzyme *maltase* digests sugar maltose. An enzyme that hydrolyzes peptide bonds of a protein is a *proteinase*, *protease*, or *peptidase*. Some fats and other lipids are digested by *lipases*. DNA is hydrolyzed by *deoxyribonuclease*, generally shortened to DNase. A *synthetase* or *polymerase* bonds together many small molecules into large molecules.

Anabolism

Any process that results in *synthesis* of cell molecules and structures. It is a building and *bond-making process* that forms *larger macromolecules from smaller ones*, and usually requires the *input of energy*.

Catabolism

Catabolic reactions *break the bonds of larger molecules into smaller molecules* and often *release energy*.

Energy in Cells (cont'd)

Cells do not create energy from nutrients; instead, they actually extract chemical energy already present in nutrient fuels and apply the energy toward useful work in the cell. Cells possess specialized enzyme systems that trap the energy present in the bonds of nutrients as they are progressively broken. During exergonic reactions, energy released by bonds is stored in certain high-energy phosphate bonds, such as in ATP. The ability of ATP to temporarily store and release the energy of chemical bonds fuels endergonic cell reactions.

Energy in Cells

Cells manage energy in the form of chemical reactions that change molecules. This often involves activities such as the making or breaking of bonds and the transfer of electrons. Some reactions release energy. They are *exergonic*: X + Y --enzyme--> Z + energy Energy of this type is available for doing cellular work. Some reactions require it to proceed. They are *endergonic*: Energy + A + B --enzyme--> C Energy of this type are used for energy transaction. In cells, exergonic and endergonic reactions are often coupled, so that released energy is immediately put to use.

Redox Reactions (fig. 7.8)

Compounds that loses electrons are *oxidized*, and the compound that receives the electrons is *reduced*. Such oxidation-reduction (redox) reactions are required for energy transformations. Important components of cellular redox reactions are *oxidoreductases, which remove electrons from one substrate and add them to another*. Their* coenzyme carriers* are nicotinamide adenine dinucleotide (*NAD*) and flavin adenine dinucleotide (*FAD*). Redox reactions always occur in *pairs*, with an *electron donor* and an *electron acceptor*. The energy present in the electron acceptor can be captured to *phosphorylate* (add an inorganic phosphate) to ADP or to some other compound. This process stores the energy in a high-energy molecule (ATP, for example). Keep in mind that hydrogens are often involved in the transfer process of electrons. The removal of hydrogens (a hydrogen atom consists of a single proton and a single elctron) from a compound during a redox reaction is called dehydrogenation. The job of handling these protons and electrons falls to one or more carriers, which function as short-term repositories for the electrons until they can be transferred.

Electron Carriers: Molecular Shuttles

Electron carriers resemble shuttles that are alternately loaded and unloaded, *repeatedly accepting and releasing electrons and hydrogens to facilitate the transfer of redox energy*. In catabolic pathways, electrons are extracted and carried through a series of redox reactions until the final electron acceptor at the end of a particular pathway is reached. In aerobic metbaolism, this acceptor is molecular oxygen, in anaerobic metabolism, it is some other inorganic or organic compound.

Enzymes

Enzymes are a special class of macromolecules. They are an example of *catalysts*, chemicals that *increase the rate of a chemical reaction without becoming part of the products* or being consumed in the reaction. Remember, enzymes don't create reactions, it speeds up a reaction.

Classification of Enzyme Functions

Enzymes are classified and named according to characteristics such as site of action, type of action, and substrate. An enzyme name is composed of a prefix or stem word derived from a certain characteristic (usually the substrate acted upon or the type of reaction catalyzed, or both) followed by the ending -ase.

Regulation of Enzyme Action (fig. 7.4)

Enzymes are not all produced in equal amounts or at equal rates. Some called *constitutive enzymes*, are always present and in relatively constant amounts, regardless of the amount of substrate. Other enzymes are *regulated enzymes*, the production of which is either turned on (induced) or turned off (repressed) in response to changes in concentration of the substrate. The activity of an enzyme is highly influenced by the cell's environment. Enzymes operate only under natural temperature, pH, and osmotic pressure of an organism's habitat. If they're subject to changes, they tend to be *labile* (unstable).Low temperatures inhibit catalysis, and high temperatures denature the apoenzyme.

Enzyme-Substrate Interactions (fig. 7.3)

For a reaction to take place, a temporary enzyme-substrate union must occur at the active site. The fit is so specific that it is often described as a "lock-and-key" fit. *Once the enzyme-substrate complex has formed, appropriate reactions occur on the substrate, often with the aid of a cofactor, and a product is formed and released*. The process can then be repeated again.

The Pursuit and Utilization of Energy

In order to carry out an array of metabolic processes, cells require constant input and expenditure of some form of usable energy. The energy comes directly from light or is contained in chemical bonds and released when substances are catabolized, or broken down. The energy is mostly stored in ATP.

Metabolic Pathways (fig. 7.5)

Metabolic reactions rarely consist of a single action or step -- they occur in a multistep series or pathway, with each step catalyzed by an enzyme. The product of one reaction is often the reactant (substrate) for the next, forming a linear chain of reactions. Others take cyclic form, in which the starting molecule is regenerated to initiate another turn of the cycle. Additionally, pathways generally do not stand alone; they are interconnected and merge at many sites.

Metabolism

Metabolism means change. It pertains to *all chemical reactions* and physical workings of the cell. There are two categories of metabolism... Anabolism and catabolism. In summary, metabolism performs these functions: 1. Assembles smaller molecules into larger macromolecules needed for the cell; in this process, ATP (energy) is utilized to form bonds (anabolism). 2. Degrades macromolecules into smaller molecules, a process which yields energy (catabolism). 3. Stores energy in the form of ATP (adenosine phosphate).

Enzyme Structure

Most enzymes are proteins -- although there is a special class that are made of RNA -- and they can be classified as simple or conjugated. *Simple* enzymes consist of *protein alone*, whereas conjugated enzymes (called *holoenzyme*) contain protein (called *apoenzyme*) and nonprotein (*cofactors*) molecules. Cofactors are either organic molecules, called *coenzymes*, or inorganic elements (metal ions). For example, catalase, an enzyme that breaks down hydrogen peroxide, requires iron as a metallic cofactor.

Controls on Enzyme Synthesis (fig. 7.7)

Some enzymes wear out, some are deliberately degraded, and others are diluted with each cell division. In order for catalysis to continue, enzymes eventually must be replaced. This cycle works into the scheme of the cell, where replacement of enzymes can be regulated according to cell demand. This system is genetic in nature -- it requires regulation of DNA and the protein synthesis machinery. *Enzyme repression* is a means to stop further synthesis of an enzyme somewhere along its pathway. As the level of the end product from a given enzymatic reaction has built to excess, the excess product will eventually bind to the DNA and shut down further enzyme production. *Enzyme induction* is reliant upon the present substrates available to the enzyme. For instance, a particular strain of E. coli is inoculated into a medium whose principal carbon source is lactose, it will then produce the enzyme lactase to hydrolyze it into glucose and galactose. If the bacterium is subsequently inoculated into a medium containing only sucrose as a carbon source, it will cease synthesizing lactase and begin synthesizing sucrose. The synthesis of an enzyme is thus induced by its substrate. The control of enzymes prevents a microbe from wasting energy.

Direct Controls on the Action of Enzymes (fig. 7.6)

The bacterial cell can slow down enzymatic activity by using two different forms of inhibition. *Competitive inhibition* is when a "mimic" substrate binds to the enzyme's active site, blocking the normal substrate from binding to the enzyme. The mimic and the normal substrate are thus competing for the active site. *Noncompetitve inhibition* occurs with special types of enzymes that have two binding sites -- the active site and the regulatory site. These enzymes are regulated by the binding of molecules other than the substrate in their regulator sites. Often the regulatory molecule is the product of the enzymatic reaction itself. This provides a negative feedback mechanism that can slow down enzymatic activity once a certain concentration of product is produced.

Cofactors: Supporting the Work of Enzymes

The metallic cofactors, including iron, copper, magnesium, manganese, zinc, cobalt, selenium, and many others, participate in precise functions between the enzyme and its substrate. In general, metals *activate enzymes*, help *bring the active site and substrate close together*, and *participate directly* in chemical reactions with the enzyme-substrate complex. *Coenzymes are a type of cofactor*. They are *organic compounds that work in conjunction with an apoenzyme to perform a necessary alteration of a substrate*. The general function of a coenzyme is to remove a chemical group from one substrate molecule and add it to another substrate, thereby serving as a transient carrier of this group. Many coenzymes are derived from *vitamins*.

Adenosine Triphosphate: Metabolic Money

The powerhouse molecule, ATP, has also been described as metabolic money because it can be earned, banked, saved, spent, and exchanged. As a temporary energy repository, ATP provides a connection between energy-yielding catabolism and the other cellular activities that require energy.