Enzymology: Enzymes as biological catalysts

• Enzymology is the study of enzymes.

Enzyme Definition

• Enzymes are biological catalysts, made of proteins or RNA, that accelerate chemical reactions.
• Enzymes lower the activation energy of a reaction without being consumed or altered in the process.
• Without enzymes, cellular chemical reactions would be too slow or not occur at all.

Activation Energy

• Activation energy is the amount of energy required to destabilize the bonds of a molecule, moving the reaction over an "energy hill".
• Catalysts reduce the amount of energy needed to start a reaction.

Enzymes as Catalysts

• Enzymes provide a surface (active site) where a substrate can bind, leading to the weakening of high-energy bonds.
• Binding holds substrates in the correct position/orientation, increasing the chance of a reaction.
• All enzymes are proteins, except for some RNAs, but not all proteins are enzymes.

Active Site of Enzymes

• The active site of an enzyme is the area where the substrate or substrates attach.
• Amino acids present in the active site play an important role in enzyme function; if one of these amino acids is altered through mutations, the enzyme could become useless.

Substrate Binding

• The simplistic model of enzyme action is that the substrate fits into 3-D structure of enzyme's active site, like "key fits into lock".
• Enzymes and substrates recognize each other, the enzyme's active site is complementary in conformation with substrate.
• The enyzme is the lock, and the reactant is the key.
• A more accurate model is the induced fit model, where the 3-D structure of the enzyme fits the substrate, and substrate binding causes the enzyme to change shape for a tighter fit due to a "conformational change".
• This brings chemical groups into position to catalyze the reaction.

Enzyme and Substrate Binding

• Bonding forces during substrate binding include ionic bonds, hydrogen bonds, and van der Waals forces.
• An active site is nearly the right shape for the substrate, and binding alters the shape of the enzyme (induced fit).
• Binding strains bonds in the substrate, involving intermolecular forces between functional groups of both the substrate and the active site.
• The active site alters its shape to maximize intermolecular bonding.

Overall Process of Enzyme Catalysis

• The active site must have strong enough binding interactions to hold the substrate long enough for the reaction to occur, but weak enough to allow the product to depart afterward, this implying a fine balance.
• Drug design involves creating molecules with stronger binding interactions that result in enzyme inhibitors, which block the active site.

Enzyme Specificity

• Enzymes have varying degrees of specificity for substrates.
• Enzymes may recognize and catalyze a single substrate, a group of similar substrates, or a particular type of bond.
• Enzymes act on a certain optical or steric isomer.
• Beta-glycosidase reacts with beta-glycosidic bonds in cellulose, while alpha-glycosidic linkages are present in starch and glycogen.

Apoenzymes and Holoenzymes

• An apoenzyme is an enzyme without its non-protein moiety and is mostly inactive.
• A holoenzyme is an enzyme with its non-protein component and is mostly active.

Compounds that Help Enzymes: Activators

• Non-protein activators may be cofactors, which are small inorganic compounds & ions such as Mg, K, Ca, Zn, Fe, and Cu that are bound within the enzyme molecule.
• Coenzymes are non-protein, organic molecules that bind temporarily or permanently to enzymes near the active site.
• Many coenyzmes are vitamins such as NAD (niacin; B3), FAD (riboflavin; B2), and Coenzyme A

Classification of Enzymes

• Enzymes are classified according to the type of reactions they catalyze.
• Oxidoreductases catalyze oxidation-reduction reactions.
• Transferases transfer groups of atoms.
• Hydrolases catalyze hydrolysis..
• Lyases add/remove atoms to/from a double bond.
• Isomerases rearrange atoms.
• Ligases use ATP to combine molecules.

Factors Affecting Enzyme Function

• Several factors influence enzyme function: enzyme concentration, substrate concentration, temperature, pH, salinity, activators, and inhibitors.

Enzyme Concentration

• As enzyme concentration increases, the reaction rate typically increases.
• With more enzymes comes more frequent collisions between enzyme and substrate, however the reaction rate eventually levels off as substrate becomes the limiting factor.
• When the substrate is the limiting factor not all enzyme molecules are able to find substrate to bind.

Enzyme Concentration & Rate of Reaction Rule

• With a constant substrate concentration [S], increases in enzyme concentration [E] lead to higher initial reaction rates (Vo).
• It is key to note that Vo is always directly proportional to the amount of enzyme present.

Substrate Concentration Rule

• With constant enzyme concentration [E], increasing amounts of substrate [S] will typically lead to a greater reaction rate.
• When reaction rate levels off, all enzymes have their active sites engaged, and the enzyme is considered saturated.

Temperature

• Each enzyme operates best at an optimum temperature, where there are greatest number of molecular collisions.
• Human enzymes typically function at 35°-40°C, with the normal body temperature at 37°C .
• Increase beyond optimum T°: Increased energy levels can disrupt bonds in enzyme and the bonds between enzyme and the susbrate, and cause denaturation (loss of 3D shape/3° structure), from weak bonds like H or ionic bonds breaking.
• Decrease in To: Molecules move slower, leading to decreased collisions between enzyme and substrate
• Temperature preferences differ with diverse enzymes, and organisms in different environments.

pH

• Changes in pH, adding or removing H+, disrupts bonds and the 3D shape due to altered attractions between charged amino acids, affecting 2° & 3° structures and causing denaturation.
• Most human enzymes function best at pH 6-8; conditions depend on localized conditions.
• Pepsin (stomach) functions at pH 2-3, trypsin (small intestines) functions at pH 8.

Salinity (Salt Concentration)

• Changes in salinity, through addition/removal of cations (+) and anions (-), disrupt bonds and the 3D shape of enzymes, altering attractions between charged amino acids and potentially resulting in denaturation.
• Key to note that enzymes are intolerant of extreme salinity.

Mechanism of Enzyme Regulation

• Enzyme activity is regulated so that product formation responds to needs of the cell.
• Regulation of enzyme activity is achieved by two general mechanisms:
  • Control of enzyme quantity (transcriptional regulation)
  • Altering the catalytic efficiency of the enzyme (allosteric regulation, covalent modification, inhibition, and Proenzyme/Zymogen)

Allosteric Regulation

• Regulatory molecules cause conformational changes.
• Allosteric inhibitors are negative effectors, and keep enzyme in inactive form
• Allosteric activators are positive effectors, and keep enzyme in active form
• The enzyme has two sites: a catalytic site for substrate binding and a regulatory allosteric site, to which an effector molecule binds.
• A binding of the allosteric effector to enzyme that increases activity has positive effector or allosteric activator.
• ADP functions as an allosteric activator for pyruvate kinase enzyme.
• A binding of the allosteric effector to enzyme that decreases activity has negative effector or allosteric inhibitor.
• ATP functions as an allosteric inhibitors for pyruvate kinase.

Feedback Inhibition

• Regulation and coordination of production where the product is used by next step in pathway.
• An example of feedbac inhibited is threonine being synthesis of isoleucine which the allosteric inhibitor of the first step in the pathway.
• With feedback inhibition there is no unnecessary accumulation of product, since final product is inhibitor of earlier step.

Proenzymes (Zymogens)

• Some enzymes are secreted in inactive forms, called proenzymes or zymogens.
• Examples include pepsinogen, trypsinogen, chymotrypsinogen, prothrombin, and clotting factors.
• Some enzymes are secreted in zymogen form to protect the tissues of origin from auto-digestion.
• A zymogen is inactive because it contains an additional polypeptide chain that masks (blocks) the active site of the enzyme.
• Activation of zymogen occurs by removal of the polypeptide chain.

Covalent Modification

• This is modification of enzyme activity through formation of covalent bonds, such as methylation (addition of methyl group), hydroxylation (addition of hydroxyl group), adenylation (addition of adenylic acid), and phosphorylation (addition of phosphate group).

Phosphorylation

• Phosphorylation is the addition of phosphate group that is the most common covalent modification used to regulate enzyme activity by addition of phosphate group to the hydroxyl group of serine, threonine or tyrosine.
• This occurs by protein kinase enzyme.
• Dephosphorylation is the removal of phosphate group from the hydroxyl group of serine, threonine or tyrosine, this occurs by phosphatase enzyme.
• The phosphorylated form is the active form in some enzymes, while the dephosphorylated form is the active form in other enzymes.

Michaelis-Menten Constant

• The Michaelis-Menten Constant is the concentration of substrate ([S]) required to half saturate the enzyme or cause half the maximal reaction rate (1/2 Vmax), this is denoted by Km
• A low Km value indicates a high affinity of enzyme for substrate, and a Low [S] is needed to half saturate the enzyme.
• A high Km value implies low affinity to the enzyme for substrate. High [S] is needed to saturate the enzyme.

The double reciprocal Plot: Lineweaver Burk plot

• The reciprocal of V is plotted versus reciprocal of [S].
• The curve is straight line.
• The slope is equal to Km/Vmax.
• More practical to estimate Km.

Enzyme Inhibition

• Inhibitors can is classified based upon their site of action on the enzyme, on whether or not they chemically modify the enzyme, or on the kinetic parameters they influence.
• Inhibitors may be either reversible or irreversible.

Competitive (Reversible) Inhibitors

• The inhibitor binds reversibly to the active site, with intermolecular bond involved.
• There is no reaction takes place on the inhibitor.
• Inhibition depends on the strength of inhibitor binding and inhibitor concentration.
• The concentration of substrate is blocked from the active site.
• Increasing substrate concentration reverses inhibition, inhibitor likely to be similar in structure to the substrate.
• An example is the "competing" of an Inhibitor & substrate with penicillin to block enzymes bacteria use to build cell walls.
• Disulfiram (Antabuse) is an example that treats chronic alcoholism by block enzymes that breaks down alcohol, resulting in severe side effects.

Non Competitive (Reversible) Allosteric inhibitors

• The inhibitor binds reversibly to the allosteric site.
• Intermolecular bonds are formed, and the induced fit alters the shape of the enzyme.
• The active site is distorted and is not recognized by the substrate.
• Increasing substrate concentration does not reverse inhibition.
• The inhibitor is not similar in structure to the substrate.

Non-Competitive Inhibitor Example

• Cyanide poisoning, which irreversibly inhibits Cytochrome C, stops the production of ATP and leads to allosteric inhibition.

Irreversible Inhibition

• Inhibitor permanently binds to enzyme.
• This permanently binds to active site.
• Permanently changes shape of enzyme to allosteric site.
• Examples include nerve gas, sarin, many insecticides, and cholinesterase inhibitors.

Isoenzymes

• These are different forms of an enzyme that catalyze the same reaction in different tissues in the body.
• They have slight variations in the amino acid sequences of the subunits of their quaternary structure.
• An example includes lactate dehydrogenase (LDH), which converts lactate to pyruvate, consists of five isoenzymes. Key is the varying tissues.

Diagnostic Enzymes

• The levels of diagnostic enzymes in the blood can be used determine the amount of damage in specific tissues.