METABOLISM and ENZYMES

Metabolism: management of materials and energy resources of the cell.

Two basic kinds of reactions:

1. Catabolic- complex molecules are broken down into simpler ones. Energy is released (an exergonic reaction) ex: cellular respiration

2. Anabolic- complex molecules are built up from simpler ones. Energy is required (an endergonic reaction) ex: protein synthesis

Energy in endergonic and exergonic reactions

For most reactions in the cell ATP is the immediate energy source.

When ATP is broken down into ADP by hydrolysis, energy is released. When ATP is formed energy is required.

Exergonic reactions occur spontaneously, but may take a long time. A catalyst is a chemical that increases the rate of the reaction without taking part in the reaction. An enzyme is a biological catalyst.

Exergonic reactions also require some energy to get started, even though the net energy will be greater. Enzymes also lower the energy of activation which is usually provided in the form of heat.

Without enzymes, considering the conditions in the cell (moderate temperature, pH, pressure), reactions would be too slow to support life.

Endergonic reactions do not happen spontaneously and require energy, again the amount of energy required is less with an enzyme

Enzymes are specific to the reactant they act on called the substrate. So we say that enzymes are substrate specific. The specificity is due to the shape of the enzyme (remember protein structure- this is the tertiary structure of the protein).

Enzymes bind to the substrate at a specific place on the enzyme molecule called the active site. This forms an enzyme-substrate complex.

While they are joined the catalytic action of the enzyme converts the reactant/s to the product/s.Example:

Maltose + water glucose + glucosemaltase

Enzymes have an active site where the catalytic activity takes place. Most often the substrate and enzyme are held together by Hydrogen bonds. The active site is made up of the R groups (providing the specificity). Once the reaction has happened, the bond is broken and the enzyme can go on to catalyze another reaction.

How do enzymes lower the energy of activation ?

1. In a reaction involving two or more reactants the enzyme provides a template for them to come together in the correct orientation.

2. The active site holds the substrates, stretching and bending critical chemical bonds that must be broken

3. The active site may provide a microenvironment that is conductive to the reaction. Ex: an aa with an acidic R group would provide a small acid pocket- this would facilitate the transfer of H+ ions to the substrate catalyzing the reaction

4. There may be a brief bonding of the R group to the active site which causes a chemical change in the substrate, inducing a reaction- the bond would break after the reaction started, making the enzyme the same again.

There are two theories about how the enzyme and substrate interact:

1.The Lock and Key theory: this theory proposes that the enzyme is like a key fitting a lock- the shapes are fixed, neither the lock or the key change their shape.

2. Induced Fit: In this hypothesis, the substrate does not simply bind with the active site. It has to bring about changes to the shape of the active site to activate the enzyme and make the reaction possible.

The hypothesis suggests that when the enzyme's active site comes into contact with the right substrate, the active site slightly changes or moulds itself around the substrate for an effective fit. This shape adjustment triggers catalysis and helps to explain why enzymes only catalyse specific reactions.

Rate of Reaction in an Enzyme controlled reaction

•The rate of reaction is measured either by the amount of reactant used up or the amount of product formed

•Under ideal conditions (which are variable) there is a maximum rate of reaction, called V max

•With constant enzyme and substrate the rate of reaction is highest at the beginning, Why?

Variables that affect enzyme activity:

1. Temperature

2. pH

3. Substrate/enzyme concentration

TEMPERATURE

Increase in temperature causes

1) More energetic collisions

2) The number of collisions per unit time will increase.

3) The heat of the molecules in the system will increase.

These will all decrease the energy of activation and speed up the reaction.

But…since we are talking about reactions in cells we have to consider the environment inside a living organism and the effect that heat has on enzymes. At a certain point the temperature will become too great and the enzyme will loose its shape and no longer be able to function. This is called denaturing.

Each enzyme has a temperature range in which a maximal rate of reaction is achieved. This

maximum is known as the temperature optimum of the enzyme.

                                                       

Enzyme in cold water shrimp

Enzyme in a bacteria living in a hot spring

Digestive enzyme in human

The amount of substrate will affect the rate of reaction- as substrate concentration increases, rate of reaction will increase proportionally until all the enzymes are saturated, then it will level off.

Substrate concentration

Increasing the amount of substrate increases the number of collisions, assuming there is enough enzyme present.

Increasing the amount of enzyme will also increase the rate of reaction, but this is limited by the amount of substrate. Enzyme is reused, substrate is not.

Graph showing effect of increasing substrate concentration

Once all enzymes are occupied, increasing substrate will no longer have an effect on rate

Effect of pH.

Each enzyme has an optimal pH, so the effect of pH will depend on the particular enzyme. Below are two proteases that work in different parts of the body. Pepsin works in the stomach, which is very acidic, Trypsin works in the small intestine where conditions are slightly alkaline.

Extremes in pH’s can also denature enzymes because their tertiary bonds will change.

Amino acid side chains contain groups such as - COOH and NH2 that readily gain or lose H+ ions.

As the pH is lowered an enzyme will tend to gain H+ ions, and eventually enough side chains will be affected so the enzyme's shape is disrupted. Likewise, as the pH is raised, the enzymes will lose H+ ions and eventually lose its active shape.

Many of the enzymes function properly in the neutral pH range and are denatured at either an extremely high or low pH.

Measuring the rate of reaction of catalase

•Catalase is present in most living cells.

•It catalyzes the breakdown of hydrogen peroxide into water and oxygen

•2H2O2 O2 + 2H20 catalase

•There are several ways to measure this reaction in the lab

•Research and present one of these protocols:

-volume of gas produced

- amount of hydrogen peroxide reacted by titrating with potassium permanganate

- time taken for catalase soaked disc to float to top of liquid

Enzymes in Biotechnology

1. Washing powders that have enzymes are called biological washing powders. They act on certain stains such as blood, grass stains:proteases, oils, fats: lipases. They make the detergent more effective (gets stains out better) and more efficient (use less)

2. Use of lactase in producing lactose-free milk

* The disaccharide lactose is present in milk and milk products.

* 70% of adults can’t breakdown lactose and so it builds up in the intestine (only monosaccharides can be absorbed)

* The bacteria that live in the gut can switch to lactose as their energy source by “turning on” the gene for lactase (an example of control of gene expression called the lac operon model).

* When the bacteria use the lactose it produces hydrogen gas and the person has intestinal symptoms.

* It is therefore benificial to have lactose-free milk available.

Production of lactose-free milk

* Milk is passed over the enzyme lactase, which is bound to an inert carrier.

* The lactose is converted to glucose and galactose, which can be absorbed

* Most milk is low in lactose but can also be made with no lactose at all (these come from plant sources like soy)

* Milk can also be treated with a bacterium like acidopholus, which is used in yogurt making.

3. Fruit juice production

Pectinases increase the yield of juice from fruit and make it clearer. Pectin is a large polysaccharide found in the cell wall. The enzymes hydrolyze the pectins and enable the easy extraction of larger volumes of clear fruit juice. Pectinase is an enzyme that is extracted from a fungus (Aspergillus niger). This fungus grows naturally on fruits and uses this enzyme to soften cell walls enabling its hyphae to grow through them.

                                                                  

                                    

Other examples of enzymes in food technology:

• Tenderizing meat with papain (a protease extracted from papaya

• Conversion of starch into sugar in brewing using amyloglucosidase

Instructions for catalase lab:

1. Fill burette with ml of potassium permanganate

2. Measure 10 ml of H2O2, 1 ml of yeast suspension and 10 ml of H2SO4

3. Mix the yeast with the H2O2 and start timing. Baseline group add 1 ml of water instead of yeast

4. Add the H2O2 at YOUR time interval. Remove 5 ml of that solution

5. Note the volume of the burette at the beginning

6. Titrate until you have a persistent pink or brown color

7. Note the end volume and subtract to find the volume used

Enzyme review

Control and inhibition of enzyme reactions

ENZYME INHIBITION

Certain chemicals can inhibit the action of an enzyme. Inhibitors work by attaching to the enzyme.

If it attaches by a covalent bond it will be a permanent inhibition because this is an irreversible process.

In some cases it can be reversed, if the bond is a weak one. There are two main kinds of inhibitors: competitive and non-competitive

Competitive inhibitors compete for the active site on the enzyme. They have a similar shape as the substrate and so block the active site so that the substrate can’t bind to the enzyme. A competitive inhibitor’s affect can be lessened if more substrate is added. If there is more substrate than inhibitor the substrate will have a better chance to gain entry to the site.

Examples of competitive inhibitors:

1. an important enzyme in the Krebs cycle (in cellular respiration) is succinate, it can be inhibited by malonate, which has a similar structure

2. Sulfa antibiotics inhibit folic acid synthesis in bacteria:

No inhibitor

Competitive inhibitor present

Graph showing affect of increasing the amount of substrate on the rate of enzymatic reaction. Given enough substrate the reaction can reach its maximum rate with a competitive inhibitor.

The antibiotic sulfanilamide is similar in structure to para-aminobenzoic acid (PABA), an intermediate in the biosynthetic pathway for folic acid. Sulfanilamide can competitively inhibit the enzyme that has PABA as it's normal substrate by competitively occupying the active site of the

enzyme.

A Non-competitive inhibitor binds somewhere other than the active site and alters the shape of the enzyme. In this case, adding more substrate will not affect the rate of reaction.

Examples: metals such as mercury, copper, silver, inhibit many enzymes because they break the disulfide bridges. Poisons such as nerve gas, and snake venom which inhibits cholinesterase, the enzyme that metabolizes ACH a neurotransmitter

Because a non-competitive inhibitor acts on a site other than the active site; increasing the substrate concentration

will not affect the rate of reaction

Metabolism in a cell occurs in metabolic pathways:

•series of chemical reactions

•requires a set of enzymes, a different one for each reaction

•each molecule that is produced is different

•each substrate is transformed into a product that serves as the substrate for the next reaction until a final product is generated called an end product

•the pathway is directional

26Theodor Hanekamp © 2003

A simple pathwaypen

cat

men

man

mat

e-a

n-t

p-m

m-c

Control of Metabolism

There has to be a system for shitting down a metabolic pathway or the cell would not only be inefficient there would be chemical chaos.

The pathways must be tightly controlled so only substances that are needed and the right amounts are produced.

This is accomplished by two ways: gene regulation and enzyme regulation.

We will look at enzyme regulation through end product inhibition.

Allosteric control of metabolism by allosteric enzymes

Molecules that regulate metabolic pathways act like reversible, non-competitive inhibitors.

They bind to a specific site on the enzyme which is remote from the active site.

Example of an allosteric enzyme with a negative effector site. When the effector molecule binds to the allosteric site, substrate binding and catalytic activity of the enzyme are inactivated. When the effector is detached from the allosteric site the enzyme is active.

Allosteric activators have the opposite effect, they will activate an enzyme by stabalizing the enzyme in the active form

Example of allosteric inhibition by an end product in a metabolic pathway

•Threonine Deaminase is the first enzyme in the metabolic pathway of changing threonine to isoeucine.•Isoleucine, the end product, can inhibit threonine deaminase•The inhibition occurs at an inhibition site on the enzyme but not the active site•An excess of end product switches off any more production of that product.•As the end product is used up it detaches from the inhibitory site.•The active site becomes active again and the pathway switches back on. Similar to non-competitive inhibition.•This mechanism makes the pathway self-regulating in terms of product manufacture--> excess product pathway shut down, product in short supply, pathway back on.

Example

In the metabolic pathway of glycolysis (the initial steps in cellular respiration where glucose is split into 2, 3 carbon molecules), there is inhibition provided by ATP (the end product).:

In one of the first steps in glycolysis

Phosphofructokinase (PFK) catalyzes a reaction. This is enzyme is allosteric and one of the main regulators of glycolysis in the cell. 

PFK is inhibited by high levels of ATP. This will stop cellular respiration if there is adequate ATP available in the cell. If there are low levels of ATP and or high levels of ATP or AMP, then the metabolic pathway is turned on.

Allosteric inhibition is an example of negative feedback.

Negative feedback is a regulatory mechanism that keeps an organism or system in dynamic balance. (like a thermostat, keeping a constant temperature in a water bath). Negative feedback will slow or stop a process, positive feedback will speed up a process. NFB maintains equilibrium, PFB causes disequilibrium. There are few examples of positive feedback in an organism but lots of negative feedback: control of glucose, temperature, etc.

Design Catalase Lab

What factors could affect the rate of reaction in the breakdown of hydrogen peroxide?

Possibly research inhibitors of catalase. Catalse from different sources

Consider levels of IV i.e, if you choose Temp, how many different temps will you use?

Consider sample size and replicates

Method of data collection for DV, other ways of measuring DV

Data analysis

Design due next class

mass

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mass

Decrease in mass vs pH

Enzyme question on protease

pH

Mass dec. (mg)