Ch. 19: Enzymes & Vitamins

Ch. 19: Enzymes & Vitamins

Catalysis by Enzymes • Enzyme: A protein that acts as a catalyst for a biochemical reaction. • Reminder: a catalyst speeds up a reaction, but at the end of the reaction is unchanged itself. The reaction rate is increased by lowering the activation energy. • Substrate: A reactant in an enzyme catalyzed reaction. • Active site: A pocket in an enzyme with the specific shape and chemical makeup necessary to bind a substrate and where the reaction takes place.

An enzyme’s activity is limited to a certain substrate and a certain type of reaction. We call this the specificity of the enzyme. • Enzymes differs greatly in their specificity. • Catalase, for example, is almost completely specific for one reaction – decomposition of hydrogen peroxide, a necessary reaction that destroys hydrogen peroxide before it damages biomolecules by oxidizing them.

Enzymes are specific with respect to stereochemistry; they catalyze reaction of only one of the pair of enantiomers. • For example, the enzyme lactate dehydrogenase catalyzes the removal of hydrogen from L-lactate but not from D-lactate.

The specificity of an enzyme for one of two enantiomers is a matter of fit. One enantiomer fits better into the active site of the enzyme than the other enantiomer. Enzyme catalyzes reaction of the enantiomer that fits better into the active site of the enzyme.

Turnover Number • The catalytic activity of an enzyme is measured by its turnover number. • turnover number: the maximum number of substrate molecules acted upon by one molecule of the enzyme per unit time. • Most enzymes have a turnover rate of 10 – 1,000 molecules per second, but some are much faster. (Catalase can turn over 10 million molecules per second…this is the fastest reaction rate you can obtain in the body because it is the rate at which molecules collide.)

Enzyme Cofactors • Many enzymes are conjugated proteins that require nonprotein portions known as cofactors. • Some cofactors are metal ions, others are nonprotein organic molecules called coenzymes. • An enzyme may require a metal-ion, a coenzyme, or both to function.

Cofactors • Cofactors provide additional chemically active functional groups which are not present in the side chains of amino acids that made up the enzyme.

Metal Ion Cofactors • Metal ions may anchor a substrate in the active site or may participate in the catalyzed reaction. • The requirement that many enzymes have for metal ion cofactors explains our dietary need for trace minerals. (The ions of Fr, Zn, Cu, MN, Ni, V, and Se all function as enzyme cofactors.) • These ions form coordinate covalent bonds by accepting lone-pair electrons present on nitrogen or oxygen atoms in enzymes or substrates.

Enzyme Classification • Enzymes are divided into six main classes according to the general kind of reaction they catalyze, and each class is further subdivided. • 1. Oxidoreductases: Catalyze oxidation-reduction reactions, most common addition or removal of oxygen or hydrogen. A– + B → A + B– In this example, A is the reductant (electron donor) and B is the oxidant (electron acceptor). In biochemical reactions, the redox reactions are sometimes more difficult to see, such as this reaction from glycolysis: • Pi + glyceraldehyde-3-phosphate + NAD+ → NADH + H+ + 1,3-bisphosphoglycerate In this reaction, NAD+ is the oxidant (electron acceptor), and glyceraldehyde-3-phosphate is the reductant (electron donor).

Enzyme Classification Cont. • 2. Transferases: Catalyze transfer of a group from one molecule to another. • A–X + B → A + B–X • In this example, A would be the donor, and B would be the acceptor. The donor is often a coenzyme. • Hydrolases: Catalyze the hydrolysis of substrate – • the breaking of bond with addition of water. • A–B + H2O → A–OH + B–H • For example, a nuclease is a hydrolase that cleaves nucleic acids.

4. Isomerases: Catalyze the isomerization (rearrangement of atoms) of a substrate in reactions that have one substrate and one product. Isomerases thus catalyze reactions of the form A → B where B is an isomer of A. 5. Lyases: Catalyze the addition of a molecule such as H2O, CO2, or NH3 to a double bond or reverse reaction in which a molecule is eliminated to create a double bond.For example, an enzyme that catalyzed this reaction would be a lyase : ATP→ cAMP + PPi (protein phosphate inhibitor) Lyases differ from other enzymes in that they only require one substrate for the reaction in one direction, but two substrates for the reverse reaction. Enzyme Classification Cont.

Enzyme Classification Cont. • 6. Ligases:Catalyze the bonding of two substrate molecules. • Ligases split C-C, C-O, C-N, C-S and C-halogen bonds without hydrolysis or oxidation. • The reaction is usually accompanied by the consumption of a • high energy compound such as ATP and other nucleoside • triphosphates. An example of this type of enzyme is pyruvate carboxylase • which catalyses the following reaction: • pyruvate + HCO3- + ATP = Oxaloacetate + ADP + Pi

Misc. Comments • Note that in our 6 examples the enzymes have their names ending in “-ase”. The modern naming always has two parts. • first part identifies the substrate on which the enzyme operates • second part is an enzme subclass name like those in Table 19.2 • Ex. pyruvate carboxylase: a ligase that acts on the substrate pyruvate to add a carboxyl group • Some enzymes are still named with older, common names (papain, trypsin) • Note that enzymes can catalyze both the forward and reverse reactions. Where both directions are of significance, the equations are often written with both arrows.

How Enzyme Work • Must explain specificity and how they lower Eact. • Specificity is determined by the active site. • Exactly the right environment for the reaction is provided in the active site. • It has the side-chain groups that attract and hold the substrate in position by intermolecular attractions and sometimes covalent bonding. • Two modes are invoked to represent the interaction between substrate and enzymes. These are: • Lock-and-key model: The substrate is described as fitting into the active site as a key fits into a lock. • Induced fit model (more modern version)

Induced-fit-model: The enzyme has a flexible active site that changes shape to accommodate the substrate and facilitate the reaction.

In enzyme catalyzed reactions, substrates are drawn into the active site to form a enzyme-substrate complex. Within the enzyme-substrate complex, the enzyme promoted reactions takes place. • Once the chemical reaction is over, enzyme separates from the substrate and restores its original conditions, becomes available for another reaction.

But What About Eact? • Before complex formation, the substrate molecule is in its most stable, lowest energy shape. • When complexed, the molecule is forces into a less stable shape and bonding electrons may be drawn away from some bonds in preparation for breaking them and forming new bonds. • The result is to lower the activation energy barrier between substrate and product.

Enzymes Act as Catalysts Because of Their Ability To: • Bring substrates and catalytic sites together (proximity effect) • Hold substrates at the exact distance and in the exact orientation necessary for reaction (orientation effect) • Provide acidic, basic or other types of groups required for catalysis (catalytic effect) • Lower the energy barrier by inducing strain in bonds in the substrate molecule (energy effect).

Effect of Concentration on Enzyme Activity • Variation in concentration of enzyme or substrate alters the rate of enzyme catalyzed reactions. • Substrate concentration: • At low substrate concentration, the reaction rate is directly proportional to the substrate concentration.(This is because not all the enzyme molecules are in use.) • With increasing substrate concentration, the rate drops off as more of the active sites are occupied. (Once the enzyme is saturated, adding more substrate has no effect on the rate).

Fig 19.5 Change of reaction rate with substrate concentration when enzyme concentration is constant.

Enzyme concentration: The reaction rate varies directly with the enzyme concentration as long as the substrate concentration does not become a limitation.

Effect of Temperature and pH on Enzyme Activity • Enzymes maximum catalytic activity are highly dependent on temperature and pH. • Increase in temperature increases the rate of enzyme catalyzed reactions. The rates reach a maximum and then begins to decrease. The decrease in rate at higher temperature is due to denaturation of enzymes.

Fig 19.7 (a) Effect of temperature on reaction rate

Denaturation • Most enzymes denature and lose their catalytic activity above 50-60 °C. • This is why medical instruments can be sterilized by heating with steam in an autoclave. • The high temperature of the steam denatures the enzymes of any bacteria present, thereby killing them.

Effect of pH on Enzyme activity: The catalytic activity of enzymes depends on pH and usually has a well defined optimum point for maximum catalytic activity.

Enzyme Regulation: Feedback and Allosteric Control • Concentration of thousands of different chemicals vary continuously in living organisms which requires regulation of enzyme activity. • Any process that starts or increase the activity of an enzyme is activation. • Any process that stops or slows the activity of an enzyme is inhibition.

Mechanisms to Control Enzyme Activity 1. Feedback control: Regulation of an enzyme’s activity by the product of a reaction later in a pathway. • biochemical reaction pathways depend on a series of consecutive reactions. • The product of one reaction is the reactant for the next. 1 2 3 A  B  C  D if D inhibits enzyme 1, it will decrease the amount of A converted to B, and the synthesis of B and C will decrease in turn.

Mechanisms Cont. 2. Allosteric control: Activity of an enzyme is controlled by the binding of an activator or inhibitor at a location other than the active site. Most allosteric enzymes have more than one protein chain and two kinds of binding sites – those for substrates and those for regulators. Binding of a regulator changes the shape of the enzyme. This alters the shape of the active site and thus affects the ability of the enzyme to bind its substrate and catalyze its reaction. Allosteric controls are further classified as positive or negative. • - A positive regulator changes the activity site so that the enzyme becomes a better catalyst and rate accelerates. • - A negative regulator changes the activity site so that the enzyme becomes less effective catalyst and rate slows down.

A positive regulator changes the activity site so that the enzyme becomes a better catalyst and rate accelerates. A negative regulator changes the activity site so that the enzyme becomes less effective catalyst and rate slows down.

Enzyme Regulation: Inhibition • The inhibition of an enzyme can be reversible or irreversible. • Inreversible inhibition, the inhibitor can leave, restoring the enzyme to its uninhibited level of activity. • Inirreversible inhibition, the inhibitor remains permanently bound to the enzyme and the enzyme is permanently inhibited.

The rates of enzyme catalyzed reactions with or without a competitive inhibitor are shown below.

Inhibitions are further classified as: • Competitive inhibitionif the inhibitor binds to the active site.

House Example • Competitive inhibition can be put to good use in treating unhealthy conditions. • Example: methanol poisoning. • methanol is harmful because it is oxidized in the body to formaldehyde which is highly toxic • ethanol has a molecular similarity to methanol, so it acts as a competitive inhibitor of the methanol dehydrogenase enzyme • with the oxidation of methanol blocked by ethanol, the methanol is excreted without causing harm • thus medical treatment of methanol poisoning includes administering ethanol

Noncompetitive inhibition, if the inhibitor binds elsewhere and not to the active site. by binding elsewhere it exerts allosteric control: changes the enzyme’s shaper so the active site is less accessible or works less efficiently

Irreversible Inhibition • The inhibitor forms covalent bonds to the active site, permanently blocking it. • Many irreversible inhibitors are poisons because of their ability to completely shut down the active site. • heavy metal ions like Hg+2 or Pb+2 • organophosphorus insecticides (parathion, malathion) • nerve gases (Sarin)

Sarin • acetylcholinesterase normally breaks down acetylcholine immediately after that molecule has transmitted a nerve impulse • Sarin irreversibly inhibits acetylcholinesterase • without this enzymes activity, acetylcholine blocks transmission of further nerve impulses, resulting in paralysis of muscle fibers and death from respiratory failure.

Enzyme Regulation: Covalent Modification and Genetic Control • Covalent modification: Two general modes of enzyme regulation by covalent modification 1. removal of a covalently bonded portion of an enzyme 2. addition of a group. Zymogens(or pro-enzymes) become active only when a chemical reaction splits off part of the molecule. • Example: 3 enzymes that digest proteins in the small intestine are produced in the pancreas as zymogens. These enzymes must be inactive when they’re synthesized so that they do not immediately digest the pancreas. The zymogens have a polypeptide segment at one end that is not present in the active enzyme. The extra segments are snipped off to make the active enzyme once they reach the small intestine where protein digestion occurs.

Ex. addition of phosphoryl groups (-PO3-2) • kinase enzymes catalyze the addition of a phosphoryl group supplied by ATP (phosphorylation) • phosphatase enzymes catalyze the removal of the phosphoryl group (dephosphorylation) • see p. 605 and discuss

Enzyme Regulation Cont. • Genetic control: The synthesis of enzymes is regulated by genes. Mechanisms controlled by hormones can accelerates or decelerates enzyme synthesis.

Summary: Mechanisms of Enzyme Control • feedback • inhibition • production of inactive enzymes (zymogens) • covalent modification of an enzyme by addition and remove of a phosphoryl group • genetic control

Vitamins • Vitamins: An organic molecule, essential in trace amounts that must be obtained in the diet because it is not synthesized in the body. • Vitamins are classified as water-soluble and fat-soluble.

Water-soluble thiamine riboflavin niacin B6 folic acid B12 biotin pantothenic acid C Fat-soluble A D E K none has been identified as a co-enzyme overdose hazard greater than for water-soluble vitamins Vitamins

Antioxidants • a substance that prevents oxidation by reacting itself with an oxidizing agent • helps protect us against active oxidizing agents that are by-products (free radicals) of normal metabolism • a free radical is an atom or molecule with an unpaired electron • free radicals quickly gain stability by picking up electrons from nearby molecules, which are thereby damaged • our main dietary ones are: vitamin C, vitamin E, B-carotene, and the mineral selenium

Chapter Summary • Enzymes are catalysts for biochemical reactions. • Enzymes are mostly water soluble and globular. • Many enzyme require a co-factor that are metal ions or non-protein organic molecules known as coenzymes. • There are six major classes of reactions catalyzed by enzymes. • Enzymes draws substrates into its active site and hold them in its active site by non-covalent interactions to produce enzyme-substrate complex.

Chapter Summary Cont. • Reactions take place within the enzyme-substrate complexes. • When the reaction is over, product is released and the enzyme returns to its original conditions. • With increasing temperature, rate of enzyme catalyzed reactions increases to a maximum and then starts to decrease as the enzyme protein denatures at high temperature. • Enzyme catalyzed reaction rate is maximal at an optimum pH.

Chapter Summary Cont. • The effectiveness of enzymes is controlled by a variety of activation and inhibition mechanisms. • Vitamins are organic molecules required in small amounts in the diet because our body can not synthesize them.

Ch. 19: Enzymes & Vitamins