The Effect of Inhibitor Ions on the Respiration of Yeast

The Effect of Inhibitor Ions on the Respiration of Yeast
The yeast to be used in the experiment is saccharomyces cerevisiae. This yeast is used in the bakery. The diagram below shows the respiration that takes place in the yeast.
The respiration in the yeast take place due to the presence of enzymes. These enzymes help in the respiration of yeast. As explained later, if the enzymes are inhibited, then the process of respiration also is inhibited. The cell membrane of Saccharomyces cerevisiae is a primary site of heavy metal toxicity by Cd2+ and Cu2+, with resultant loss of mobile cellular solutes, such as K+. Silver, in addition to loss of K+, has been reported to increase efflux of accumulated phosphate, mannitol, succinate, glutamine, and proline. Mercury and silver both inhibit yeast respiration. A specific target for mercury has not been defined, but atp content of the cell is rapidly depleted. Silver is reported to bind with phosphate, resulting in collapse of the proton motive force. Toxic metal ions, including Cu2+, Co2+, Ni2+, Cd2+, Mn2+, and Hg2+, also inhibit plasma membrane ATPase by means of various binding interactions. Silver and mercury have relatively high affinities for reduced thiol groups, but which of the many thiol-containing cellular constituents, such as glutathione, cysteine, or coenzyme A, and thiol-containing proteins are affected. Definition of an enzyme An enzyme is a biological catalyst. A catalyst is a chemical that can lower the activation energy required for a chemical reaction to take place, which in turn increases the rate of reaction. Enzymes are unique in that they are highly specific in their action, catalysing only one or a small range of reactions. An enzyme can make it easier for two molecules to join together, or for a molecule to be split into two new products. Without enzymes, chemical reactions in our cells would not take place nearly as fast as they are required to. Enzymes are therefore essential for maintaining a metabolic rate fast enough to sustain life. How an enzyme functions In a traditional, non-enzyme controlled reaction, the molecules need to collide with sufficient force (kinetic energy) for the bonds to be broken / made. Not only do they need to collide with sufficient force, they need to hit each other in the correct positions for any reactions to occur. This means the process is highly inefficient and requires a large amount of energy. The diagram below illustrates this: [image] Diagram from ?Introduction to Advanced Biology? By CJ Clegg, 2000 John Murray Enzymes overcome this problem. Instead of having the molecules collide and react, the molecules attach to the enzyme at an area known as the active site. This forms what is known as an enzyme-substrate complex. This large molecule then breaks down to form the broken down products of the initial molecule (known as the substrate molecule) or a product created by the bonding of two substrate molecules. As I have stated in my definition, enzymes are highly specific. This is because the active site of an enzyme is an inverse copy of the shape of the substrate molecules it is designed for. This means no other substrate molecules are able to join with the enzyme molecule. This idea of enzyme ? substrate rigidity is known as the lock and key hypothesis, meaning only the correct ?key? (substrate) will work the ?lock? (enzyme.) The diagram below shows how the enzyme will only accept certain substrate molecules. Only molecule E is the correct shape. [image] The enzyme does not however, stay in one shape. It is flexible, allowing the substrate to manoeuvre into place before taking its shape for catalysing the substrate. This movement is caused by chemical reactions and ionic repulsion and attraction between the two / three molecules. This is known as the induced fit theory. [image] Diagram ? 1994 Encyclopaedia Britannica Inc. At step A, the enzyme molecule is not anything like the shape of the substrate. At step B, The enzyme molecule has been induced to change its shape by the electrostatic forces created by the presence of the substrate. It now fits the substrate perfectly. At step C, The reaction has been completed, as the substrate has been broken down into its products. The enzyme will now release the products Inhibitors Certain molecules can inhibit enzyme action. The inhibition of enzyme action is not rare or purely of academic significance. Organisms use inhibitors to control the rate at which metabolic reactions in the body take place. Inhibitors work by binding tightly to the enzyme, destroying its catalytic properties. Inhibitors can be Non-reversible, or reversible. This means they will either permanently stop enzyme action, or will be a temporary obstacle, displaced by other substrate molecules. Inhibitors can also be classed as competitive, or non-competitive. Competitive inhibitors compete with substrate molecules for entry to the active site. Once the inhibitor has been accepted into the active site (it is the correct shape to go in) it then blocks that enzyme off to any other substrate molecules. However increasing substrate concentration will cause more collisions with substrate molecules, displacing the inhibitor. Non-competitive inhibitors work by attaching themselves to the enzyme, but not actually within the active site. The attached molecules alter the structure of the bonds of the enzyme and thus change its shape, making it unable to accept substrate molecules. In some cases, non-competitive inhibitors partially obscure the active site, making them inaccessible to substrate molecules. Examples of an irreversible inhibitor include cyanide (which reacts with cytochrome oxidase in the mitochondria and halts respiration,) and malathion, an inhibitor used in pesticides to kill insects. Heavy metals are also effective enzyme inhibitors. The graph below shows how the different inhibitors affect enzyme concentration: [image] Graph from ?Introduction to Advanced Biology? By CJ Clegg, 2000 John Murray Heavy metals are metals with a density beyond 5 g/cm3, thus, the transition elements from V (but not Sc and Ti) to the half-metal As, from Zr (but not Y) to Sb, from La to Po, the Lanthanides and the Actinides can be referred to as ?heavy metals?. Of the 90 naturally occuring elements, 21 are non metals, 16 are light metals and the remaining 53 (with As included) are heavy metals. Although they comprise the major part of the elements, the understanding of the metabolism of heavy metals and the biotechnological use of these metabolic functions are in their infancy. Most heavy metals are transition elements with incompletely filled d-orbitals. These d-orbitals provide heavy metal cations with the ability to form complex compounds which may be redox active or not. Thus, heavy metal cations play an important role in sophisticated biochemical reactions such as nitrogen fixation, water cleavage during oxygenic photosynthesis, respiration with oxygen or nitrate, one-electron catalysis, re-arrangement of C-C bonds, hydrogen assimilation, cleavage of urea, transcription of genes into mRNA, and programmed development of a single cell to a human being. These are all based on the formation of or catalysis by biochemical heavy metal complex compounds. At higher concentrations, however, heavy metal ions form unspecific complex compounds in the cell which lead to toxic effects. Some heavy metal cations, e. g. Hg2+, Cd2+ and Ag+, are so toxic complex-formers that they are too dangerous for any biological function. Even highly reputable trace elements like Zn2+ or Ni2+ and especially Cu2+ are toxic at higher concentrations. Thus, the intracellular concentration of heavy metal ions has to be tightly controlled, and heavy metal resistance is just a specific case of the general demand of every living cell for some heavy metal homoeostasis system. Factors affecting enzyme activity There are many factors that affect the catalysing action of enzymes. My investigation is directed only at the effect inhibitors have on enzyme molecules. Because of this, all other factors will be kept the same throughout the experiment to avoid introducing other variables and wrong results. Factors that affect the rate of an enzyme-controlled reaction are: - Temperature - pH of Solution - Substrate concentration - Enzyme concentration - Presence of an inhibitor To maximise the rate of reaction, I will keep my yeast (and therefore the enzymes) at 37 degrees centigrade by means of a water bath. This temperature has been chosen as it is the optimum temperature for the enzymes to operate at. Beyond this point, although the number of collisions with the active site is increased (the kinetic theory), the temperature causes the enzyme to denature (change shape) and therefore become ineffective The enzymes used in this experiment To investigate the affect of inhibitors on enzyme activity, a measurable product must be created as a product of the reaction. This product must be separable from other chemicals in order that its mass or volume can be recorded. In this investigation, the respiration of yeast was seen as a good solution. Yeast respire, producing CO2 gas. As respiration is an enzyme controlled series of reactions, introducing an inhibitor will affect the rate of respiration, and thus the volume of gas collected. Yeast also respire both aerobically and anaerobically. This is useful as the reaction will not stop if there is no air in the dough which the yeast are suspended in. Initially mitochondria were to be used, but the process of extraction was deemed too complicated for the limited time available. Aim The aim of this experiment is to observe how enzyme inhibitors affect the catalysing properties of enzymes (enzyme action,) using inhibitors for enzymes involved in the respiration of yeast (saccharamomyces cerevisiae.) The yeast used in this experiment is commonly used in the bakery.

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