Enzymes Prof Dr: Olfat Shaker Department of Medical Biochemistry
Introduction• Enzymes are Proteins but in some cases they are RNA e.g.snurps (ribozymes).• They are Catalysts produce by living cells, can work outside these cells.• They Speed reactions without changing the equilibrium point.• They denatured at high temp
• They function in minute amounts, remain unchanged chemically during the reaction.• Enzymes are highly specific.• They are sensitive to changes in pH and temperature.• They lose some of their activity during the reaction
Turnover numberThe “turnover number” is the number ofsubstrate molecules converted into product byan enzyme molecule in a unit time when theenzyme is fully saturated.
Chemical nature of Enzymes1. Simple proteins: only protein chain(s)2. Conjugated proteins (Holoenzyme): consists of Protein part (apoenzyme) Non-protein part : may be Organic (coenzymes): loosely attached to apoenzyme Inorganic (activator): loosely attached to apoenzyme *Prosthetic group is a non-protein part firmly attached to the apoprotein
Enzyme Terminology• Hydrolyzing enzymes are named by adding –ase to the name of substrate – sucrase – reacts sucrose – lipase - reacts lipid• Enzymes named by adding their function to the name of the substrate – Lactate dehydrogenase – remove hydrogen from lactic acid• Pepsin and trypsin
Localization of the enzyme• Intracellular enzymes :they act inside the cells that make them.• Extracellular: they act outside the cells that secret them such as: • digestive enzymes in the GIT • coagulation enzymes in plasma.
Zymogens• Digestive and coagulation enzymes are secreted in inactive forms, zymogens or proenzyme.• Zymogens are activated by trimming of a short peptide blocking the active site, or by covalent modification of the zymogen.
Structure of the enzyme• The enzyme is tertiary or quaternary structure that has spatial configuration.• It has pockets on its surface. Each pocket has its own function. –The catalytic or active site. –The allosteric site.
The catalytic or active site It is the site at which the substratebinds to the enzyme. It should be fit to the substrate(fitness is made by the tertiarystructure of the enzyme molecule). Any factor affecting this structurewill alter the fitness and formation ofenzyme-substrate complex.
The Allosteric site It is a site for fitting of a small molecule whosebinding alters the affinity of the catalytic site to thesubstrate. This small molecule is called allosteric modifier stimulatory (making it more fit) inhibitory (making the catalytic site unfit) for binding of the substrate.
Mode of enzyme action• The reactants should raise their energy levels to reach a transition state.• The transition state represents the energy barrier between the reactants and products.• This energy is known as energy of activation.• The enzyme makes the reaction needs lower activation energy.
Factors affecting enzyme action• The activity of the enzyme is evaluated by measuring the rate or the velocity of the reaction.• velocity of the reaction is measured by how many moles of the substrate are converted into products per unit of time (minute).
Factors affecting Enzyme activity • Substrate concentration • Temperature • pH • Time • Cofactors • Enzyme Inhibitors
Substrate ConcentrationAs substrateconcentration increases,• the rate of reaction increases (at constant enzyme concentration).• the enzyme eventually becomes saturated giving maximum activity.
Michaelis constant (Km)• Km is equal to the substrate concentration [S] at which the reaction is half of its maximum (½Vmax).• It expresses the affinity of the enzyme to its substrate.• Low Km means high affinity of the enzyme to the substrate• High Km means low affinity of the enzyme to the substrate
Enzymes – Lineweaver-Burk Equation V = Vmax [S] [S] + Km Inverting the Equation yields: 1 = Km 1 + 1 . (Lineweaver-Burke Equation) V Vmax [S] Vmax By plotting 1/ V as a function of 1/[S], a linear plot is obtained: Slope = Km/Vmax y-intercept = 1/Vmax
Michaelis-Menton Equation: Lineweaver-Burk Equation: Comparison of these two methods of plotting the same data:
TemperatureEnzymes• are most active at an optimum temperature (usually 37° in humans). C• show little activity at low temperatures.• lose activity at high temperatures as denaturation occurs.
pHEnzymes• are most active at optimum pH.e.g: • Pepsin is 2.0 • Trypsin is 6.0. • Alkaline phosphatase is 9.5• lose activity in low or high pH as it undergoes Denaturation.
TimeAs time is passed the rate of the enzyme-catalyzed reaction diminishes due to: –Decline of substrate concentration –The accumulated product may cause feed- back inhibition of the enzyme
Cofactors• Most enzymes are associated with non-protein part which may be: metal-ion as zinc, copper, iron prosthetic group as flavine nucleotide, biotin, 4’- phophopantetheine (of pantothenic acid), or cobamide. coenzyme as nicotinamide nucleotide, NAD or NADP• All these cannot be isolated from the protein part of the enzyme (apo- enzyme) by dialysis.• The protein part of the enzyme (apo-enzyme) + [metal, prosthetic group, or the coenzyme] = holoenzyme
Inhibitors (I)Inhibition may be reversible or irreversible.• Reversible: If the inhibitor binds non-covalently to the enzyme• Irreversible: If the inhibitor binds covalently to the enzyme If [S] is > [I], only part of enzyme molecules are occupied by the [I] and the inhibition will be proportionate to [I]. [I] increases Km, so, more substrate is needed to reach Vmax.
Reversible Inhibitors Competitive inhibitors: compete with the substrate for the same active site (catalytic site) Non-competitive inhibitors: bind to the enzyme in a location other than the active site Allosteric inhibitors: bind to the enzyme in the allosteric site, produce conformational change leading to enzyme inhibition.
Competitive inhibitors• If [I] is > [S], E-I complex is formed, and it fails to dissociate. So, inhibition of the E-S complex reaction takes place.• Examples of competitive inhibitors: • Allopurinol competes with hypoxanthine for xanthine oxidase inhibiting the formation of uric acid, so it is used in treatment of hyperuricemia (gout). • Dicumarol or Warfarine compete with vitamin K, for epoxide reductase, so they are used to reduce prothrombin synthesis. • Statins (e.g. atorvastatin) competes with HMGCoA for its reductase, so, it inhibits cholesterol synthesis. • Methotrexate competes with dihydrofolic acid for dihydrofolate reductase, so, it inhibits DNA synthesis and used in treatment of cancers.
Non-competitive inhibitors• The inhibitor binds to the enzyme in site away from the catalytic site.• Inhibition cannot be reverted by increasing [S].• Km is not changed because the inhibitor does not interfere with E-S formation, but Vmax is, apparently reduced.
Allosteric inhibitors• Allosteric inhibitors are low-molecular weight substances that bind the allosteric sites, on the surface of the enzyme.• This interaction causes conformational changes in the catalytic site that makes it unfavorable for binding to substrate.• The importance of allosteric inhibition is to avoid accumulation of the final product of succession of reactions (metabolic pathway).• End- product of a metabolic pathway inhibits the initial enzyme in the pathway, this called feed-back inhibition.
Irreversible enzyme InhibitorExamples: Lead binds to ferrochelatase during heme synthesis. Thiol group blocking agents, e.g. Iodoacetate inhibits glyceradehyde-3-P dehydrogenase Heavy metal bridges two –SH groups making Enz- S-Metal-S-Enz, Oxidizing agents that oxidize –SH into disulfide (-S- S-) Protein precipitants as alkaloidal reagents, e.g. tungestic acid denturating agents, e.g. Heating, conc. acids or alkalies.
Regulation of Enzyme activity1. Covalent modification:(short term regulation) The enzyme may be phosphorylated in the OH group of serine, threonine, or tyrosine. This process is catalyzed by kinase. ATP is the source of phosphate. The phosphoprotein produced is dephosphorylated by phosphatase. phosphoprotein enzyme is the active form of the enzyme, e.g. glycogen phosphorylase. the dephosphorylated form is the active form of the enzyme, e.g. glycogen synthase.
2. Induction and repression of enzyme synthesisEnzymes synthesis (long term regulation ).• Synthesis is stimulated by "induction" or inhibited by "repression".• Control of enzyme synthesis is under hormonal control that affects gene expression related to that particular enzyme.• Example: • Insulin induces synthesis of glucokinase and phosphofructokinase, but represses glucose-6-phosphatase and fructose 1,6,bisphosphatase. • Steroid and thyroid hormones effects on enzyme generation.• Control of enzyme activity by induction or repression consumes hours or days to be achieved.3. Allosteric modification (short term regulation) occurs within seconds or minutes.
Plasma enzymes• The only functioning plasma enzymes are those of blood coagulation secreted from the liver as zymogens.• The plasma enzymes are diffused from tissues into plasma due to turnover of tissue cells or due to diffusion of these enzymes out of the diseased cells (those undergoing degenration or involved in inflammation).• Plasma enzymes lack tissue specificity, because the same enzyme is mostly secreted from more than one tissue type.
• Examples of plasma enzymes – Lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) are present in RBCs, liver, myocardium, and skeletal muscle. – Creatine kinase in present in muscle, heart, and brain.• Their rise in plasma is not, by-itself, diagnostic to a disease. They may be of value to follow-up a disease already diagnosed.• Isoenzymes (Isozymes)• When the enzyme is a quaternary structure, some of its subunits are not identical according to tissue of origin. However, all perform the same catalytic function. They can be differentiate by electrophoresis.• Examples: • LDH has five isozymes • CK has three isozymes • alk.phosphatase has three isozymes.• Isozymes, accordingly, move differently in electrophoresis field.