Download Enzymes A simulation of Invertase Activity

Enzymes: Invertase Activity – a computer simulation
(The following text is modified from the Student Lab Manual for Biology Labs Online.)
Introduction – Enzyme Activity:
The cells that make up an animal body are complex chemical factories, breaking down and
building up molecules important for the maintenance of life. The sum total of these reactions is
referred to as Metabolism – with the breakdown, energy yielding, reactions called catabolism
and the building reactions referred to as anabolic. Often a breakdown, energy yielding reaction
is spontaneous i.e. runs downhill while anabolic (uphill) reactions must be coupled to a reaction
that releases more energy than is used by that building reaction in order for it to take place.
However, even when reactions are spontaneous, they often do not occur at a rate sufficient to
maintain life. Thus some sort of catalyst, a rate enhancing molecule is needed to promote life
functions. This entity is the protein based enzyme.
Enzymes can catalyze (accelerate) very specific chemical reactions with incredible precision
because of the specific shape assumed by the enzyme protein. The specific sequence of amino
acids that compose a protein cause that protein to fold into a discrete molecular conformation or
shape. All enzymes have a conformation that produces and active site – a pocket or groove in its
surface where its “substrate” (the reactive chemical) binds. The active site of an enzyme is
typically specific for only one substrate, because the overall molecular three-dimensional
conformation of the active site is designed to fit the molecular shape of its particular substrate.
This is often referred to as a “lock and key” arrangement, though it is more likely that the
enzyme molds itself around the substrate as it enters the pocket – this is called an “induced fit”
mechanism and provides for a certain degree of functional regulation.
Substrate binding to the active site of an enzyme is only the first step toward catalyzing a
reaction. If enzymes function as biological catalysts, then how can we determine and measure the
efficiency and rate of any reaction that a particular enzyme is carrying out on a given substrate
that it binds? To begin, it is important to remember that one key aspect of enzyme activity is that
while an enzyme speeds up the rate of a reaction, it is not altered by the reaction itself. An
enzyme does not become part of the reactants or products. After an enzyme has catalyzed a
reaction, it releases its substrate and then the active site of the enzyme is available to bind to
another fresh substrate and repeat this process. For most enzymes this process, known as the
catalytic cycle of enzyme activity, can be repeated very rapidly as long as there is enough
substrate for the enzyme to react upon. Regardless of the type of reaction an enzyme is
catalyzing–for example, a synthesis reaction or a degradation reaction–the catalytic cycle of an
enzyme is often described by the following equation:
E + S  ES  E + P
This cycle begins when an enzyme (E) binds to a substrate (S) to form an enzyme-substrate
complex (ES). Enzyme-substrate complexes typically form as a result of weak bonds between
amino acids in the active site and atoms of the substrate. Depending on the type of reaction
catalyzed by the enzyme, the enzyme then “manipulates” the substrate into the proper
conformation to catalyze a reaction – a process called “straining a bond’ into the “transition
state”. As the strained “energized” bond relaxes the final reaction occurs, for example, breaking
bonds by hydrolysis, catalyzing the formation of new bonds, or rearranging atoms in the
substrate to convert the substrate into a new molecule or molecules called products (P). Once the
reaction has occurred, the enzyme releases the product(s); thus, the active site is free and
available to bind to another substrate so the enzyme can repeat this cycle. Enzyme-catalyzed
reactions can be reversible or irreversible.
Through this cycle, enzymes are said to lower the activation energy of a reaction – the energy
required to make or break chemical bonds in a substrate to initiate a reaction. It is convenient to
think about activation energy as a barrier that a cell must overcome to enable a reaction to take
place. In order to understand why this is necessary for living cells, think about the factors that
can regulate the rate of most reactions that you might carry out in a test tube in a chemistry lab.
Increasing the temperature of the tube as well as increasing reactant concentration in the tube are
two conditions that will increase the rate of most chemical reactions. Increasing temperature
raises kinetic energy of reactants in the tube, thus increasing the likelihood of collisions between
molecules. Raising reactant concentration results in more frequent collisions between molecules.
Both factors typically increase the rate of a chemical reaction. Although heating or cooling a test
tube is an effective way to regulate enzyme activity in a controlled laboratory environment, body
temperature homeostasis prevents living organisms from raising and lowering body temperature
to accommodate the large number of reactions that occur simultaneously in any given cell.
Similarly, substrate concentrations for many biochemical reactions in a living cell are very low–
in the nanomolar to picomolar range or lower–thus, it simply is not efficient for a cell to wait for
kinetic energy to cause molecules to collide randomly and react with each other.
Think of enzymes as a set of "molecular hands" for a living cell. Consider the simple analogy of
a bag of separate nuts and bolts. If screwing one nut onto one bolt is the reaction you want to
carry out, you could expend a lot of energy shaking the bag until, over time, one nut randomly
finds its way onto the end of a bolt! The energy you expended to begin to put a nut and bolt
together is activation energy. Alternatively, you could speed up this reaction by reaching into the
bag and using your hands to screw a nut onto a bolt, thus reducing the amount of energy required
for this reaction to occur and greatly increasing the rate of this reaction.
Although enzymes are absolutely essential for accelerating biochemical reactions, a number of
conditions influence enzyme activity. Enzymes don’t always operate at their maximal rate,
however. Most enzymes demonstrate temperature and pH optimums - a temperature and pH at
which enzyme activity is greatest. For example, as you might expect, blood enzymes perform
optimally at a pH close to 7.4, the pH of normal human blood, whereas stomach enzymes have
an optimal pH of around 2.0 to coincide with acidic conditions in the stomach. Varying these
conditions typically affects the conformation of the enzyme, which in turn influences an
enzyme’s ability to bind to its substrate and catalyze a reaction. Recall that when any protein
unfolds, it becomes less active or inactive; this process is called protein denaturation. Enzymes
can become denatured in response to an increase in temperature because raising temperature can
break bonds–such as hydrogen bonds, Van der Waals attractions, and disulfide bridges–that are
responsible for the secondary and tertiary structure of a protein. Changes in pH can also disrupt
protein structure by changing hydrogen bond and ionic bond interactions, and by changing side
group (R-group) interactions that contribute to the tertiary structure of an enzyme. In the most
extreme circumstances, changing temperature or pH too dramatically – for example, by boiling–
can completely denature an enzyme, causing it to lose all of its activity.
Cell metabolism is dependent on the combined actions of many different enzymes that are
essential for the anabolic and catabolic reactions that must occur to maintain the physiology of a
cell. It is important to understand that many reactions that require enzymes rarely involve just a
single enzyme that works by an all-or-none process. This would be like trying to manufacture a
car from all of its components in one sweeping motion! Instead, many biochemical reactions
involve cascades of enzymatic reactions, called metabolic pathways, which serve to catalyze a
series of reactions. In a metabolic pathway, several enzymes work in a sequential fashion to
convert reactants into a product or products. At each step in a metabolic pathway, intermediate
molecules (metabolites) are produced that serve as the substrates for subsequent enzymes in the
pathway. One benefit of a metabolic pathway compared to a single-enzyme reaction is that a cell
can often precisely regulate the amount of product it can generate by independently controlling
the catalytic activity of certain enzymes in the pathway.
Intermediate molecules are often an important part of the control of a metabolic pathway. One
way in which metabolic pathways can be regulated by intermediates involves feedback
inhibition. In this regulatory process, a final or end-product of a reaction can inhibit enzymes in
the metabolic pathway. This process allows a cell to carefully control the amount of end-product
that it produces as a way to prevent excess accumulation and waste of an end-product.
Frequently, the end-product inhibits or blocks the activity of an enzyme at one of the initial, ratelimiting steps in the pathway to prevent the unnecessary production of intermediates. This would
be similar to a car manufacturer, whose car supply has exceeded the public’s demand, stopping
the auto assembly line at the first step rather than halfway through the assembly process to avoid
producing half-completed cars.
For the purpose of studying cell metabolism, as well as for medical treatment and other
applications, we can use molecules as enzyme inhibitors to artificially regulate enzyme activity.
Some of these inhibitors can bind to enzymes in a reversible or an irreversible fashion. The two
primary classes of enzyme inhibitors are called competitive and noncompetitive inhibitors.
Competitive inhibitors are molecules that decrease an enzyme’s activity by binding to the active
site of the enzyme and preventing binding of the enzyme’s normal substrate. Competitive
inhibitors can work in this fashion because their molecular shape so closely resembles the natural
substrate that the inhibitor is able to "compete" with the substrate for binding to the enzyme’s
active site. Although competitive inhibitor molecules bind to the active site of an enzyme,
catalysis does not occur.
Noncompetitive inhibitors also reduce or block an enzyme’s activity, but unlike competitive
inhibitors these molecules do so by binding to a portion of the protein other than the active site.
This binding results in a change in the overall three-dimensional conformation of the protein –
altering the active site so that it will not bind to the substrate.
Because many enzymes are subject to inhibition by inhibitor molecules, biologists can take
advantage of this property by designing compounds that can be used to modify enzyme activity
for the purpose of treating human ailments and disease, as well as a number of other applications.
For example, one of the more widely known competitive inhibitors is the antibiotic penicillin,
which functions to block the active site of an enzyme required for cell wall synthesis by bacteria.
Another example of a commonly used inhibitor is aspirin, which functions as a noncompetitive
inhibitor of an enzyme involved in the production of prostaglandins, molecules that cause fever
and inflammation. Last, two drugs employed in the treatment of acquired immunodeficiency
syndrome (AIDS) function to inhibit enzymes in the human immunodeficiency virus (HIV). The
first anti-HIV drug developed for patient use, AZT (3'-azido-2,'3'-dideoxythymidine),
competitively inhibits an enzyme required for the synthesis of HIV DNA. Another class of
enzymes, called protease inhibitors, function to reduce replication of HIV by competitive
inhibition of an essential viral enzyme called HIV protease.
Invertase:
Now that you are familiar with some important biochemical properties of enzymes, it is time to
put your knowledge to work. Countless numbers of different enzymes exhibit many of the
properties discussed in this background. In EnzymeLab you will work with an enzyme that most
likely played an important role in digesting some of the food molecules that you ate this morning
for breakfast! The enzyme chosen for this lab is Invertase (b -fructofuranosidase; E.C. 3.2.1.26),
also commonly called sucrase and saccharase. This enzyme uses water to catalyze the hydrolysis
of the disaccharide sucrose. Sucrose, or "table sugar," is technically called a -D-glucopyranosyl
(1  2) b -D-fructofuranoside because it consists of the two simple sugars glucose (a -Dglucopyranosyl) and fructose (b -D-fructofuranoside) joined together by a glycosidic bond
connecting carbon 1 of glucose to carbon 2 of the fructose molecule. Invertase cleaves the
glycosidic bond between glucose and fructose. In animals, this reaction is required to digest
sucrose and release the monosaccharides glucose and fructose to make them readily available for
absorption into the bloodstream. This reaction is important because most organisms cannot
metabolize sucrose directly–it must first be converted to monosaccharides for cells to utilize
sucrose as an energy source.
Invertase is present in a wide range of organisms including animals, plants, yeast, fungi, and
algae. In humans, invertase is found on the surface of epithelial cells lining the inner walls of the
small intestine. Depending on the source of invertase, the enzyme is active at a range of
temperatures from 40° C to 70° C. Invertase can also be active in the pH range from 4.0 to 10.0.
Two of your goals for this laboratory are to determine the optimal temperature and pH for the
invertase you will study in EnzymeLab. You should be aware that this computer simulation
exercise is possible for Invertase, since the amino acids forming the active site and the way these
amino acids interact with and bind to the substrate have been well characterized in extensive
previous “wet-lab” studies.
Objectives:
You will use EnzymeLab Simulation to study important biochemical
parameters of enzyme-catalyzed reactions as illustrated by Invertase.
You will set up an experiment by adding substrate to a test tube along with
purified enzyme to determine:
 The effects of changes in substrate concentration
 The temperature and pH optimums for invertase
 The effect of inhibitors on invertase activity.
Computer modeling is a significant, and growing, element of scientific
research for everything from molecular ecology (genetics), to meteorology and
astrophysics.
In most cases, models allow scientists to conduct experiments that would be
very difficult to conduct otherwise. This could be due to the complexity
(molecular ecology), the size of the data set involved (meteorology), or just the
impossibility of doing the experiment in real life (galactic motion). Obviously,
the models scientists use are not just games, or random number generators!
Models are generated based on the knowledge learned from previous
research, and carefully programmed to produce the most realistic, and
meaningful data possible. Otherwise, they would not be useful!
For this teaching laboratory, we are utilizing a sophisticated model based on
the understanding of enzymes generated over the last century to conduct
experiments that would be difficult to manage in the limited time we have in
class. I suspect that last week’s lab probably gave you first-hand experience
with how complicated these experiments would be to do in lab!
Once you enter the simulation follow the instructions listed under the
“Assignments for non-majors”.