Enzymes

Enzyme Characteristics all Enzymes are Proteins all Enzymes are Catalysts – i.e. control the rate of a chemical reaction

How Enzymes work Enzymes bind and hold substrates (aka reactants) in a certain orientation to speed the chemical reaction along Enzymes change shape as they bind the substrates

the binding substrates + enzyme-substrate complex active site

the reaction, the release enzyme-substrate complex product

What about the other way? substrate enzyme-substrate complex enzyme product

Lactase 1926 aa’s long cell membranes - small intestines Lactase

Beano - alpha galactosidase breaks down trisaccharides raffinose – in beans, cabbage enzyme not in humans in bacteria in large intestines Raffinose is a trisaccharide composed of galactose, fructose, and glucose. It can be found in beans, cabbage, brussels sprouts, broccoli, asparagus, other vegetables, and whole grains. Raffinose can be hydrolyzed to D-galactose and sucrose by the enzyme α-galactosidase (α-GAL), an enzyme not found in the human digestive tract. Humans and other monogastric animals (pigs and poultry) do not possess the α-GAL enzyme to break down RFOs and these oligosaccharides pass undigested through the stomach and upper intestine. In the lower intestine, they are fermented by gas-producing bacteria which do possess the α-GAL enzyme and make carbon dioxide, methane, and/or hydrogen—leading to the flatulence commonly associated with eating beans and other vegetables. α-GAL is present in digestive aids such as the product Beano. Beano is a product containing the enzyme alpha galactosidase, which is derived from the fungus Aspergillus niger. +

another example – glyceraldehyde-3-dehydrogenase

re-introducing activation energy activation energy is the energy required to get a reaction going activation energy net energy change

How do Enzymes work? They lower the “activation energy” of the reaction activation energy is the energy required to get a reaction going activation energy Enzymes increase the likelihood that the molecules will collide successfully if two are to be joined. They also can increase the stress on a bond, increasing the likelihood that it will break. net energy change

How do they do it? They lower the “activation energy” of the reaction net energy change

Enzymes Concentration and Reaction Rates Here we show two different set ups for enzymes. One set up has 2 enzymes and the second has 3 fold more or 6. We let the reaction run and count the number of substrate molecules present every twn seconds. Then we graphed that data. At first the number of product molecules created increases in a linear manner, but with time their rate of accumulating slows down. Why? Because it’s harder for the enzyme to find substrate. The rate at which product accumulates is quicker and plateaus sooner for the higher concentration of enzyme – as you would expect. More enzymes means more product quicker.

Enzyme performance is affected by: Concentration of substrates present Temperature pH Inhibitors Poisons

Substrate Concentration and Reaction Rates Now we use the same on line application to look at how reaction rates change when you keep the amount of enzyme constant but you change the amount of substrate available to be acted on by the enzyme. As expected, with more substrate available, the enzymes have a greater chance of colliding and interacting and the reaction rate, the number of product molecules made per second, increases.

Substrate Concentration and Reaction Rates When we talk about enzymes we usually are talking about their reaction rate. Every enzyme has a maximum reaction rate, a maximum speed, - that is how fast it can catalyze the reaction. To get a sense of how fast an enzyme can be, we measure the amount of product that accumulates over time for several different concentrations of substrate. We have created a plot of this information using data from the on line application. As expected, the more substrate that is available, the faster product appears, to a point. That point is the point at which all the enzymes are busy. So doing the experiment with even more substrate does make the product appear any faster. We can identify the maximum reaction rate by creating a second curve using the initial slopes of the product versus time graphs. When we plot the values of the slopes versus the substrate concentrations, we see that the resulting curve flattens out much like the growth curves we studied in ecology where the populations reached the ecosystem’s carrying capacity. By identifying the y value of the plateau in the second graph, we get the maximum reaction rate for that enzyme at that concentration of the enzyme. Once we have the reaction rate, we can use this to study how other factors like temperature and pH affect enzyme reaction rate.

Enzymes and Temperature temperature  reaction rate because increased kinetic energy breaks H-bonds temperature  reaction rate because decreased kinetic energy does not break H-bonds Enzymes are temperature sensitive and have an optimal temperature at which they operate the fastest. When doing a enzyme temperature experiment, the rate of reaction for a fixed amount of substrate and enzyme is calculated based on best fit graphs at various temperatures. That data is then plotted as reaction rate (enzyme activity) versus temperature. A best fit curve of the data indicates the enzyme’s maximal temperature.

pH Acids – excess Hydrogen ions Bases – excess hydroxyl ions Neutral – equal numbers of H+ and OH-

Enzymes and pH each enzyme has an optimal pH; some work best in acidic conditions (<4) (pepsin) while others work best closer to a neutral pH (7) pH for Optimum Activity Enzyme pH Optimum Lipase (pancreas) 8.0 Lipase (stomach) 4.0 - 5.0 Lipase (castor oil) 4.7 Pepsin 1.5 - 1.6 Trypsin 7.8 - 8.7 Urease 7.0 Invertase 4.5 Maltase 6.1 - 6.8 Amylase (pancreas) 6.7 - 7.0 Amylase (malt) 4.6 - 5.2 Catalase 7.0

Enzymes and Inhibitors A number of factors can degrade enzyme activity. There are two categories – non-specific factors that work on a wide variety of enzymes i.e. they are not enzyme specific, and specific inhibitors that work on specific enzymes. Non specific inhibitors usually cause the enzyme to “denature” i.e. change shape so it no longer works. Specific inhibitors work by binding to the enzyme. That means they have a shape that “fits” on some part of the enzyme. As they bind they may irreversibly change the shape – this is irreversible inhibition. Or they make bind loosely so that they can fall away. This is reversible inhibition. Of the types of reversible inhibition, the inhibitor may compete for the active site (competitive inhibition) or may bind away from the active site but still alter the active site (noncompetitive inhibition).

Competitive Inhibitors With competitive inhibitors, the competing molecule competes for the active site. When it is occupying the active site, the enzyme cannot catalyse the reaction. It must wait until the inhibitor leaves before it can resume catalyzing reactions. How do you think the reaction rate is changed in the presence of a competitive inhibitor?

Noncompetitive Inhibitors A noncompetitive inhibitor is a substance that interacts with the enzyme, but usually not at the active site. The noncompetitive inhibitor reacts either remote from or very close to the active site. The net effect of a non competitive inhibitor is to change the shape of the enzyme and thus the active site, so that the substrate can no longer interact with the enzyme to give a reaction. Non competitive inhibitors are usually reversible, but are not influenced by concentrations of the substrate as is the case for a reversible competitive inhibitor. See the graphic on the left. Irreversible Inhibitors form strong covalent bonds with an enzyme. These inhibitors may act at, near, or remote from the active site. Consequently, they may not be displaced by the addition of excess substrate. In any case, the basic structure of the enzyme is modified to the degree that it ceases to work. Since many enzymes contain sulfhydral (-SH), alcohol, or acid groups as part of their active sites, any chemical which can react with them acts as an irreversible inhibitor. Heavy metals such as Ag+, Hg2+, Pb2+ have strong affinities for -SH groups. Nerve gases such as diisopropylfluorophosphate (DFP) inhibit the active site of acetylcholine esterase by reacting with the hydroxyl group of serine to make an ester. Oxalic and citric acid inhibit blood clotting by forming complexes with calcium ions necessary for the enzyme metal ion activator.

Enzymes and Inhibitors bind to specific enzymes and decrease the reaction rate Normal substrate enzyme binding Competitive inhibitor binds to the active site Noncompetitive inhibitor binds to the enzyme and changes its shape

Poisons - KCN Irreversible Inhibitor of Cytochrome C Oxidase, ATP cannot be made Anaerobic respiration only Fatal build up - Lactic Acid

Poisons - Arsenic Reversible nonspecific Inhibitor of pyruvate dehydrogenase Glucose cannot be broken down Cell death results