Enzymes: Introduction Enzymes are macromolecules that catalyze chemical reactions in the body. Most enzymes are very specific – they catalyze only one particular reaction.

What Are Enzymes? Enzymes are large molecules that increase the rates of chemical reactions without themselves undergoing any change. The all known enzymes are globular proteins. However, proteins are not the only biological catalysts

Catalyst substance that increase rates of a chemical reaction does not effect equilibrium remain unchanged in overall process reactants bind to catalyst, products are released

Catalytic Power  Enzymes can accelerate reactions as much as over uncatalyzed rates!  Urease is a good example: –Catalyzed rate: 3x10 4 /sec –Uncatalyzed rate: 3x /sec –Ratio is 1x10 14 !

trypsin, an enzyme that cleaves the peptide bonds of protein molecules-but not every peptide bond, only those on the carboxyl side of lysine and arginine residues. The enzyme carboxypeptidase specifically catalyzes the hydrolysis on only the last amino acid on a protein chain-the one at the C- terminal end. Lipases are less specific: They catalyze the hydrolysis of any triglyceride, but they still do not affect carbohydrates or proteins.

How Are Enzymes Named and Classified? Enzymes are commonly given names derived from the reaction that they catalyze and/or the compound or type of compound on which they act lactate dehydrogenase speeds up the removal of hydrogen from lactate (an oxidation reaction). Acid phosphatase catalyzes the hydrolysis of phosphate ester bonds under acidic conditions

Enzymes can be classified into six major groups according to the type of reaction they catalyze 1- Oxidoreductases catalyze oxidations and reductions. 2- Transferases catalyze the transfer of a group of atoms, such as from one molecule to another. 3- Hydrolases catalyze hydrolysis reactions. 4- Lyases catalyze the addition of two groups to a double bond or the removal of two groups from adjacent atoms to create a double bond. 5- Isomerases catalyze isomerization reactions. 6- Ligases, or synthetases, catalyze the joining of two molecules.

What Is the Terminology Used with Enzymes?  Some enzymes, such as pepsin and trypsin, consist of polypeptide chains only.  Other enzymes contain nonprotein portions called cofactors (co - enzyme).

Co-enzymes Non-protein molecules that help enzymes function Associate with active site of enzyme Enzyme + Co-enzyme = holoenzyme Enzyme alone = apoenzyme Organic co-enzymes – thiamin, riboflavin, niacin, biotin Inorganic co-enzymes – Mg ++, Fe ++, Zn ++, Mn ++

Substrate : is the compound on which the enzyme works, and whose reaction it speeds up. The substrate usually binds to the enzyme surface while it undergoes the reaction. The active site. The substrate binds to a specific portion of the enzyme during the reaction, If the enzyme has coenzymes, they are located at the active site. Therefore, the substrate is simultaneously surrounded by parts of the apoenzyme, coenzyme, and metal ion cofactor

Activation is any process that initiates or increases the action of an enzyme. It can be the simple addition of a cofactor to an apoenzyme or the cleavage of a polypeptide chain of a proenzyme. Inhibition is the opposite-any process that makes an active enzyme less active or inactive. Inhibitors are compounds that accomplish this task. Competitive inhibitors bind to the active site of the enzyme surface, thereby preventing the binding of substrate. Noncompetitive inhibitors, which bind to some other portion of the enzyme surface, may sufficiently alter the tertiary structure of the enzyme so that its catalytic effectiveness is reduced. Both competitive and noncompetitive inhibition are reversible, but some compounds alter the structure of the enzyme permanently and thus make it irreversibly inactive.

What Factors Influence Enzyme Activity? Enzyme activity is a measure of how much reaction rates are increased.

A. Enzyme and Substrate Concentration If we keep the concentration of substrate constant and increase the concentration of enzyme, the rate increases linearly. That is, if the enzyme concentration doubles, the rate doubles as well; if the enzyme concentration triples, the rate also triples. This is the case in practically all enzyme reactions, because the molar concentration of enzyme is almost always much lower than that of substrate.

 Conversely, if we keep the concentration of enzyme constant and increase the concentration of substrate, we get an entirely different type of curve, called a saturation curve.

 At the saturation point, substrate molecules are bound to all available active sites of the enzymes. Because the reactions take place at the active sites. once they are all occupied, the reaction is proceeding at its maximum rate.  Increasing the substrate concentration can no longer increase the rate because the excess substrate cannot find any active sites to which to bind.

B. Temperature Temperature affects enzyme activity because it changes the conformation of the enzyme.  In uncatalyzed reactions, the rate usually increases as the temperature increases.  Changing the temperature has a different effect on enzyme-catalyzed reactions.

When we start at a low temperature, an increase in temperature first causes an increase in rate. However, protein conformations are very sensitive to temperature changes. Once the optimal temperature is reached, any further increase in temperature alters the enzyme conformation. The substrate may then not fit properly onto the changed enzyme surface, so the rate of reaction actually decreases.

 After a small temperature increase above the optimum, the decreased rate could be increased again by lowering the temperature because, over a narrow temperature range, changes in conformation are reversible.  However, at some higher temperature above the optimum, we reach a point where the protein denatures; the conformation is then altered irreversibly, and the polypeptide chain cannot refold to its native conformation. At this point, the enzyme is completely inactivated. The inactivation of enzymes at low temperatures is used in the preservation of food by refrigeration.

Most enzymes from bacteria and higher organisms have an optimal temperature around 37°C. However, the enzymes of organisms that live at the ocean floor at 2°C have an optimal temperature in that range.

C.pH As the pH of its environment changes the conformation of a protein, Each enzyme operates best at a certain pH.  Once again, within a narrow pH range, changes in enzyme activity are reversible.  However, at extreme pH values, enzymes are denatured irreversibly, and enzyme activity cannot be restored by changing back to the optimal pH.

What Are the Mechanisms of Enzyme Action? The action of enzymes is highly specific for a substrate. What kind of mechanism can account for such specificity?  About 100 years ago, Arrhenius suggested that catalysts speed up reactions by combining with the substrate to form some kind of intermediate compound.  In an enzyme-catalyzed reaction, this intermediate is called the enzyme ­substrate complex.

To account for the high substrate specificity of most enzyme-catalyzed reactions, a number of models have been proposed.

A. Lock-and-Key Model This model assumes that the enzyme is a rigid, three-dimensional body. The surface that contains the active site has a restricted opening into which only one kind of substrate can fit, just as only the proper key can fit exactly into a lock and turn it open.

According to the lock-and-key model, an enzyme molecule has its particular shape because that shape is necessary to maintain the active site in exactly the conformation required for that particular reaction.

An enzyme molecule is very large (typically consisting of 100 to 200 amino acid residues), but the active site is usually composed of only two or a few amino acid residues, which may well be located at different places in the chain. This arrangement emphasizes that the shape and the functional groups on the surface of the active site are of utmost importance in recognizing a substrate. The other amino acids-those that are not part of the active site-are located in the sequence in which we find them because that sequence causes the molecule as a whole to fold up in exactly the required way.

B. induced – Fit Model From x-ray diffraction, we know that the size and shape of the active site cavity change when the substrate enters. To explain this phenomenon, an American biochemist, Daniel Koshland, introduced the induced-fit model, in which the compared the changes occurring in the shape of the cavity upon substrate binding to the changes in the shape of a glove when a hand is inserted. That is, the enzyme modifies the shape of the active site to accommodate the substrate.

C. Catalytic Power of Enzymes  Both the lock-and-key model and the induced-fit model emphasize the shape of the active site. However, the chemistry at the active site is actually the most important factor.  A survey of known active sites of enzymes shows that five amino acids participate in the active sites in more than 65% of all cases. They are, in order of their dominance, His> Cys > Asp> Arg > Glu., reveals that most of these amino acids have either acidic or basic side chains. Thus acid-base chemistry often underlies the mode of catalysis.

In any reaction that can be written as follows: A + B → C + D before A and B can become C and D, they must pass through a transition state where they are something in between. This situation is often thought of as being an "energy hill" that must be scaled. The energy required to climb this hill is the activation energy.

Enzymes are powerful catalysts because they lower the energy hill. They reduce the activation energy.

How the enzyme reduces the activation energy is very specific to the enzyme and the reaction being catalyzed. The specific amino acids in the active site and their exact orientation make it possible for the substrate(s) to bind to the active site and then react to form products.

Home Work Suggest a reason why enzymes can be partially protected from thermal denaturation by high concentrations of substrate.

How Are Enzymes Regulated? A.Feedback Control is an enzyme regulation process in which formation of a product inhibits an earlier reaction in the sequence.

For example, in such a system, each step is catalyzed by a different enzyme: The last product in the chain, D, may inhibit the activity of enzyme E 1 (by competitive, noncompetitive, or some other type of inhibition). When the concentration of D is low, all three reactions proceed rapidly. As the concentration of D increases, however, the action of E 1 becomes inhibited and eventually stops. In this manner, the accumulation of D serves as a message that tells enzyme E 1 to shut down because the cell has enough D for its present needs. Shutting down E 1 stops the entire process.

B. Proenzymes Some enzymes are manufactured by the body in an inactive form. To make them active, a small part of their polypeptide chain must be removed. These inactive forms of enzymes are called proenzymes or zymogens. After the excess polypeptide chain is removed, the enzyme becomes active.

For example, trypsin is manufactured as the inactive molecule trypsinogen (a zymogen). When a fragment containing six amino acid residues is removed from the N- terminal end, the molecule becomes a fully active trypsin molecule. Removal of the fragment not only shortens the chain; but also changes the three- dimensional structure (the tertiary structure), thereby allowing the molecule to achieve its active form.

C. Allosterism Sometimes regulation takes place by means of an event that occurs at a site other than the active site but that eventually affects the active site. This type of interaction is called allosterism, and any enzyme regulated by this mechanism is called an allosteric enzyme.

If a substance binds noncovalently and reversibly to a site other than the active site. it may affect the enzyme in either of two ways:  negative modulation: inhibit enzyme action  positive modulation: stimulate enzyme action

o The substance that binds to the allosteric enzyme is called a regulator, o and the site to which it attaches is called a regulatory site. In most cases, allosteric enzymes contain more than one polypeptide chain (subunits); the regulatory site is on one polypeptide chain and the active site is on another.

D. Protein Modification The modification is usually a change in the primary structure, typically by addition of a functional group covalently bound to the apoenzyme. The best-known example of protein modification is the activation or inhibition of enzymes by phosphorylation.  A phosphate group is often bonded to a serine or tyrosine residue. In some enzymes, such as glycogen phosphorylase, the phosphorylated form is the active form of the enzyme. Without it, the enzyme would be less active or inactive.

 The opposite example is the enzyme pyruvate kinase.  When the activity of PK is not needed, it is phosphorylated (to PKP) by a protein kinase using ATP as a substrate as well as a source of energy.  When the system wants to turn on PK activity, the phosphate group, Pi, is removed by another enzyme, phosphatase, which renders PK active.

E. Isoenzymes Another type of regulation of enzyme activity occurs when the same enzyme appears in different forms in different tissues. Lactate dehydrogenase (LDH) catalyzes the oxidation of lactate to pyruvate, and vice versa.  The enzyme has four subunits (tetramer). Two kinds of subunits, called H and M.  The enzyme that dominates in the heart is an H 4 enzyme, meaning that all four subunits are of the H type, although some M-type sub units are present as well.  In the liver and skeletal muscles, the M type dominates. Other types of tetramer combinations exist in different tissues: H 3 M, H 2 M 2, and HM 3. These different forms of the same enzyme are called isozymes or isoenzymes.

The different subunits confer subtle, yet important differences to the function of the enzyme in relation to the tissue. The heart is a purely aerobic organ. LDH is used to convert lactate to pyruvate in the heart. The H 4 enzyme is allosterically inhibited by high levels of pyruvate (its product) and has a higher affinity for lactate (its substrate) than does the M 4 enzyme, which is optimized for the opposite reaction. The M 4 isozyme favors the production of lactate.