II.  Overview of Chemical Reactions.

    A + B → C

where A and B are the starting materials (reactants) and C is the result (product) of this reaction. The completion and direction of this reaction depends on the free energy (G) of the system. Free energy is the energy available to do work.

A. Exergonic Reactions

A + B →  C + energy (heat, ATP)

Note that:

1. these reactions release energy;
2. the products have less energy than reactants;
3. the energy that is released during the reaction can be lost as heat or trapped in other molecules such as ATP;
4. the free energy change that occurs during this reaction, symbolized by ΔG, is equal to the difference between the energy of the products and the energy state of the reactants. In an exergonic reaction, Δ G is negative;
5. exergonic reactions are typically breakdown (hydrolytic, catabolic) reactions, such as the hydrolysis of starch or cellular respiration; and
6. exergonic reactions are spontaneous; that is, they don't need outside help (an external energy source) to occur.

Hill Model - rocks roll down a hill

B. Endergonic Reactions

A + B + energy →  C

Note that:

1. these reactions require energy;
2. the products have a greater energy content than the reactants;
3. there is a positive ΔG;
4. endergonic reactions are synthetic (buildup, anabolic) like photosynthesis; and
5. are not spontaneous (i.e., require an external energy source to occur).

    If endergonic reactions are not spontaneous, how does the cell complete these vital processes? Answer - by coupling them to an exergonic reaction so that the sum of the ΔG's for the two reactions is negative. The hydrolysis of ATP is the most common source of energy for endergonic reactions (ATP + H20 →  ADP + P; ΔG = -7.3 kcal/mol).

Example:

A + B →  C (ΔG  = +4.0 kcal/mol)
ATP + H₂0 →  ADP + P (ΔG = -7.3 kcal/mol)
A + B + ATP + H₂0 →  C + ADP + P (ΔG = -3.3 kcal/mol).

Note that since the overall ΔG is negative, this reaction sequence is now spontaneous and will occur.

The actual "coupling" process is usually the result of ATP binding to one of the starting materials. This essentially "energizes the reaction" making it thermodynamically favorable. For more, check out the notes on energetics.

C. Activation energy.
    Even if a spontaneous reaction or reaction sequence has a negative ΔG, it may only occur very slowly at the temperatures of life. For example: the conversion of bread (starch) to glucose takes about 1000 years if it sits in water. However, if you heat up the mix, it takes about 1 hour. But, eat some bread and our body converts it to glucose in minutes.

    Similarly, rocks don't just roll down hill. Most just sit at the top waiting patiently, until we give them a little push. This push is analogous to what the biochemists call activation energy (symbolized E_a), which is the minimum energy needed to start a chemical reaction. Thus, reactions require a "push", or more appropriately, the necessary activation energy to proceed.

    The Hill Model once again.
 

III. Molecules and energy
    To better understand this process, let's study the energy distribution of a population of molecules by plotting the following graph:            
                # of molecules vs. energy/molecule

    Our graph is a bell-shaped curve. We observe that some molecules have a lot of energy, some little, but most are happily average. For most reactions at life temperatures, only a fraction of the molecules have the necessary E_a - so the reaction proceeds slowly.
 

IV. What can be done to speed things up?

1. Increase the concentration of the reactants.
       Not too feasible, especially in organisms where many cell components are present only at very low concentration.
    
2. Heat.
       Works great for chemists, but not biologists. Why? 'cuz heat increases the energy of molecules, which can lead to thermal instability. Just ask a fried egg. Note we're not changing E_a, just the energy possessed by individual molecules.
    
3. Lower the Activation Energy Requirement by adding a catalyst.
       This is molecular version of the "if you can't beat ‘em, join 'em" philosophy. A catalyst is a substance that speeds up the rate of a chemical reaction by lowering the activation energy requirement for a reaction. The catalyst is not "used up" during the process, it is recovered unchanged. Once the activation energy requirement has been lowered, most of the molecules will have the necessary energy to react. This is the approach taken by life.
    

V. Enzymes - the catalysts of life

A.  General

• are catalysts (speed up a reaction, but are not consumed in the process). Thus, enzymes are not destroyed in the reactions they are involved and can be used over and over.

• are proteins, which in turn, are made of chains of amino acids linked together by peptide bonds. Remember that proteins have many different functions in organisms - enzymes, the catalytic proteins, are just one type. However, they are arguably the most important group of proteins, because without enzymes, no metabolism could occur

• are mostly globular proteins (tertiary structure) and some have quaternary structure.

• lower the activation energy requirement for a reaction

• end in "ase": ex., tyrosinASE

• named by a standardized system of nomenclature. The official name is based upon: (a) the normal substrate and (b) the type of reaction. This system was developed by the International Union of Biochemists in 1956. Enzymes may also have a trivial or common name (such as catalase).

• are usually specific, in vivo, for one reaction

• have a unique 3-D shape that is determined by the kind and sequence of amino acids in the protein

• were first studied by Jon Jakob Berzelius (1835). The Swede Arrehenius developed the concept of the enzyme-substrate complex (1889). The lock and key model was described in 1894.

B.  Mechanism of Action.
    Enzymes interact with its reactant(s), called a substrate, to form an enzyme-substrate complex. The substrate fits into an active site (catalytic region of the enzyme). The product is released and the enzyme is recovered unchanged. A brief equation: E + S →  E-S complex →  E + P. This reaction is reversible, depending upon the chemical equilibrium that is established.

    Here's some examples: catalase is an enzyme that hydrolyzes hydrogen peroxide (substrate) to oxygen and water (products).  Click here for Spuds McSaupe.  In a lab during a past semester we studied the enzyme tyrosinase.  It is found in many organisms and its main job is to oxidize (breakdown) tyrosine (one of the amino acids) and other phenolic molecules (with a benzene ring and attached hydroxyl group). Unlike many enzymes that are substrate-specific, tyrosinase isn't and works on a variety of phenolics. We took advantage of that fact and used a different substrate, L-DOPA (dihydroxyphenylalanine). Tyrosinase converts L-DOPA into the product, L-DOPA-quinone. Thus the reaction can be diagrammed:

     L-DOPA + tyrosinase →  L-DOPA-tyrosinase complex →  L-DOPA quinone + tyrosinase

C. Kinetics of a enzymatic reaction.
    Consider a typical enzyme-catalyzed reaction such as mixing catalase and hydrogen peroxide. Assuming a constant amount of enzyme, how will the concentration of the product, substrate and E-S complex change over time? At time "0", the amount of substrate is high, the product is low (zero), and the ES complex is zero. How do you predict the concentration of the product (oxygen and water), substrate (hydrogen peroxide) and ES complex to change as the reaction proceeds?

graph: concentration vs. time
 

D.  Enzymes and Substrates.
    An analogy (model) for the interaction of substrate and enzyme at the active site is the Lock-and-Key Model. The active site of the enzyme had a specific shape, like a lock, that is only recognized or opened by only a single key, like a substrate molecule. This explains why most enzymes are substrate-specific, that is, they act on only a single substrate (or sometimes a group of related substrates like tyrosinase). Now also know that the active site is not rigid like a metal lock, but that the enzyme shape is more flexible, like, say, a bean bag chair. The chair has a particular shape but it can change slightly. When you sit in it, it conforms to your body. Another good analogy is a handshake. This is called the induced fit model, the binding of the enzyme causes the active site to conform to the substrate.

E. So how do enzymes lower activation energy requirements?

1. Enzymes increase the concentration of substrates in vicinity of the enzyme;
2. Enzymes orient substrates to the most favorable position for reaction (remember for any molecules to react they must first come in contact with one another;
3. Enzymes change conformation that may strain bonds of the substrate;
4. Enzymes provide a micro-environment that may be more favorable chemically (i.e., hydrophilic/hydrophobic or acid/etc.) for reaction.
    

VI.  A Model.
    Let's use a model to help explain enzyme activity. Consider the game of musical chairs. The chair symbolize the enzymes and the seat represents the active site. The players are the substrates who will be converted into product (which happens when you sit in the seat). We will blindfold the players and allow them to wander randomly around the room (cell). [Note - the chair should also be moving, perhaps on rollers] When a player and chair collide, the player sits and is converted to product and then stands back up.

    Let's play! We'll start with 5 chairs (enzymes) and 100 players (substrates) in a large room. Every minute we will count the number of people who have contacted and sat in a chair (turned into product). Then, we will plot [product] vs. time (min).

graph: product formed vs. time

Thus, it’s obvious that enzymes can really speed up the rate of a reaction!

VII.  Factors that influence the rate of enzymatic reactions.

A. Enzyme concentration.
    For a constant amount of substrate - the more enzyme, the more active sites, the more product formed per unit time (the greater the reaction rate). Chair model: to simulate the effect of enzyme concentration on reaction rate, let's start with 2 chairs (enzymes) and 100 players (substrates) in a large room. Then every minute for an hour we'll count the number of people who found and sat in a chair. We can then plot the [product] vs. time Repeat with 5, 10, and 100 chairs (enzymes).

graph: product formed vs. time (for several enzyme concentrations)

We can now calculate the slope of each line to give us the reaction rate ([product formed]/minute) for each enzyme concentration. Now, we can plot reaction rate vs. enzyme concentration.

graph: reaction rate vs. enzyme concentration

Conclusion: The more chairs, the less time it will take any one person to encounter one. The more enzyme, the more active sites, the greater the reaction rate.

B. Substrate Concentration.
    For a constant enzyme concentration - as the substrate concentration increases, the rate of reaction increases until a saturation level of substrate is reached at which point no matter how much more is added, there will be no additional increase in rate. Chair model: start with 10 chairs and 10 players. Every minute for one hour record the number of players (substrates) who find and sit in a chair (enzyme). Plot the product formed (number of people sitting vs. time). Repeat with 20, 50 and 100 players.
                    graph: product formed vs. time (for several substrate concentrations

Now, calculate the slope of each line to yield a reaction rate and then plot reaction rate vs. substrate concentration.
                   
                    graph: reaction rate vs. substrate concentration

Conclusions: As the players increase, the likelihood of rapidly finding a chair increases. So the rate goes up. At low substrate concentrations, the rate-limiting step is finding a chair. At higher concentrations, the rate limiting step is the speed of conversion of enzyme into product.

    (These last three paragraphs are not on the exam) Two measures of the effectiveness of an enzyme-catalyzed reaction are Vmax and Km. Vmax = maximum rate of reaction. Km = substrate concentration that yields half the maximum reaction rate. These can be found graphically. Km for many enzymes range from 1 mM to 0.1 uM.

    Based on the relationship between reaction rate and substrate concentration in the graph above, Michaelis and Menton derived the following equation:

v = Vmax [S]/ Km + [S]

    The beauty of this equation is that if we know the substrate concentration for a given enzyme we can calculate its predicted velocity. In practice it can be difficult to determine Km and Vmax from the graph. However, using the Michaelis-Menten equation, Lineweaver and Burk realized that if a graph of 1/V vs 1/[S] is plotted, the X intercept is the negative inverse of Km and the Y intercept yields the inverse of velocity. One advantage of making a "double reciprocal" plot is that the result is a straight line and that Km and Vmax are easily to determine.

C. Temperature
    As temperature increases, so does the reaction rate because you heat the molecules and increase the probability of a collision. Each enzyme has a unique temperature optimum. For example, thermophilic bacteria can tolerate extremely hot temperatures in contrast to other organisms. The rate declines rapidly after the optimum because the enzyme denatures - looses it normal (native) shape, which in turn changes the shape of the active site making it less favorable for the reaction. Chair model: Imagine 10 chairs and 100 players. Play the game at room temperature. Repeat a 0 C and 30 and 100 C. Imagine everything moving slow at the cold temperatures. And, it's none too pleasant at 100. Hmmmm…the model doesn't work too well for this one!

D. pH.
    Each enzyme has a unique temperature optimum. A pH greater or less than the optimum result in reduced rates of reaction. pH causes changes in the structure of the enzyme (affects pH sensitive bonds that hold the enzyme in its 3D shape), and hence, changes the shape of the active site. Thus, the reaction becomes more or less favorable for the reaction to occur.

E. Enzyme Inhibitors.
    There are two groups of molecules that inhibit chemical reactions: competitive and non-competitive.

1. Competitive inhibitors.
    These compete with the substrate for the active site. These inhibitors typically have a chemical structure similar to the normal substrate. Some examples: (a) succinic dehydrogenase converts succinic acid to fumaric acid. Malonic acid is a competitive inhibitor; (b) ribulose bisphosphate carboxylase binds carbon dioxide to RuBP. Oxygen is a competitive inhibitor.

2. Non-competitive Inhibitors.
    These molecules bind to a site on the enzyme other than the active site. Binding causes changes in the shape (conformation) of the enzyme, which in turn, changes the shape of the active site, and hence activity. Examples: (a) Hg and disulfide bonds (Mad Hatters and mercury)

F. Enzymatic control of metabolism

1. Allosteric effectors.
    These bind to a site (allosteric site) other than active site and cause a change (slight) in the active site. These changes will enhance or inhibit the reaction. These are used to regulate rate of metabolic processes.

2.  Availability of cofactors/coenzymes.
    These are inorganic and organic substances, respectively, that are required by enzyme, in addition to the substrate, for activity. Vitamins are important coenzymes. Many metal ions are enzyme cofactors. Cofactors and coenzymes may be bound loosely or tightly to the enzyme.

3.  Presence of activation factors.
    Some enzymes require activation. For example, pepsinogen must be converted to pepsin. Many enzymes in photosynthesis are light activated.

4. Cooperativity
    The binding of one substrate to the enzyme enhances the ability of the enzyme to bind to additional substrates.

5.  Feedback inhibition - an end product inhibits an enzyme in its pathway

6.  Multi-enzyme complexes and membrane compartmentalization.

VIII. The importance of enzymes (not tested directly on exam)

A. Many diseases can be detected by screening blood serum for enzymes. Some examples:

• Serum glutamic oxaloacetic transaminase (SGOT) is found in tissues of high metabolic activity (heart, liver, pancreas, muscle) and is released following injury. Thus it is a good indicator for myocardial infarction (MI, heart attack) and liver disease (cirrhosis, hepatitis);
• Serum glutamic pyruvic transaminase (SGPT) is found in liver, heart, muscle, kidney and is especially diagnostic for liver disease;
• Lactic dehydrogenase (LDH), is found in a wide variety of tissues, and indicates cell death and leakage. It is used to confirm a diagnosis of MI. There are five 5 isozymes: 1,2 = MI, 3=pulmonary, 5=liver;
• Creatine phosphokinase (CPK) is found in heart and skeletal muscle and is released upon injury to the myocardium and skeletal muscle;
• Acid phosphatase - which has a wide distribution, esp. liver, spleen, kidney, red blood cells, and prostate; and
• Alkaline phosphatase - is found in bone, liver, and placenta functions best pH 9, and indicates liver/bone disease.

B. Several human diseases are caused by a deficiency in a key enzyme in a biochemical pathway.
    In other words, these conditions are the result of abnormal metabolic activity. Examples include phenylketonuria and albinism.

C. A Case Study.
    Several years ago I had a complete blood work-up that included tests for SGOT and CPK. My CPK level was astronomically high, but the SGOT level was normal. My health was normal. How do you explain these results?

D. Some Take-Home-Lessons:
    (1) Certain enzymes can serve as clinical markers for some human disorders; (2) Some enzymes have a restricted distribution in an individual (i.e., found in a specific tissue, organ, cell and even organelle); (3) Enzyme deficiencies are inherited disorders (remember last semester); (4) A final, most excellent, conclusion - Properly functioning enzymes are crucial for survival.

VIII.  Commercially important enzymes include (not tested directly on exam)

• amylase - brewing - convert starch to fermentable sugar
• invertase - artificial honey - convert sucrose to glucose & fructose
• lactase - ice cream - prevent crystallization of lactose
• naringinase - citrus - remove bitter tasting naringin
• pectic enzymes - coffee - coat of bean
• pectic enzymes - juice - improve juice yield
• pectic enzymes - wine - clarification
• proteases - brewing - aid filtration & clarification
• rennet - cheese - coagulate casein
• catalase - milk - destruction of H₂O₂
• proteases - detergents - laundry aid
• photography - recovery silver from spent film
  

IX.  Some fun enzymes (not tested directly on exam)

A. Rennin
    Breaks certain bonds in casein, the major protein in milk converting it to paracasein. Paracasein is insoluble and precipitates out of solution, clotting or curdling the milk. In vivo, the function of this enzyme is to slow the passage of milk proteins through the digestive tract to improve digestion. Rennin used in cheese making was traditionally obtained from the fourth stomach of a calf or other bovine (hoofed) animal, but now it is obtained primarily from microbial fermentations (trade name of rennilase).

B. Amylase.
    Catalyzes the hydrolysis of starch to maltose. Starch is a polymer of glucose; maltose is a disaccharide of glucose. Amylase occurs in the saliva where it serves to initiate the breakdown of dietary starch. It is also abundant in seeds where it is used for breaking down starch to be used for germination.

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