Structure and physical-chemical properties of enzymes.

Accelerate reactions by a millions fold Enzymes - catalysts of biological reactions

1. Catalyze only thermodynamically possible reactions 2. Are not used or changed during the reaction. 3. Don’t change the position of equilibrium and direction of the reaction 4. Usually act by forming a transient complex with the reactant, thus stabilizing the transition state Common features for enzymes and inorganic catalysts:

Specific features of enzymes: 1. Accelerate reactions in much higher degree than inorganic catalysts 2. Specificity of action 3. Sensitivity to temperature 4. Sensitivity to pH

Structure of enzymes Enzymes Complex or holoenzymes (protein part and nonprotein part – cofactor) Simple (only protein) Apoenzyme (protein part) Cofactor Prosthetic groups -usually small inorganic molecule or atom; -usually tightly bound to apoenzyme Coenzyme -large organic molecule -loosely bound to apoenzyme

Example of prosthetic group Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc, copper, cobalt). Example of metalloenzyme: carbonic anhydrase contains zinc

Active site of lysozym consists of six amino acid residues which are far apart in sequence

Coenzymes Coenzymes act as group-transfer reagents Hydrogen, electrons, or groups of atoms can be transferred Coenzyme classification (1) Metabolite coenzymes - synthesized from common metabolites (2) Vitamin-derived coenzymes - derivatives of vitamins Vitamins cannot be synthesized by mammals, but must be obtained as nutrients

Examples of metabolite coenzymes ATP S-adenosylmethionine ATP can donate phosphoryl group S-adenosylmethionine donates methyl groups in many biosynthesis reactions

Cofactor of nitric oxide synthase 5,6,7,8 - Tetrahydrobiopterin

Vitamin-Derived Coenzymes Vitamins are required for coenzyme synthesis and must be obtained from nutrients Most vitamins must be enzymatically transformed to the coenzyme Deficit of vitamin and as result correspondent coenzyme results in the disease

Nicotinic acid (niacin) an nicotinamide are precursor of NAD and NADP Lack of niacin causes the disease pellagra NAD + and NADP + NAD and NADP are coenzymes for dehydro- genases

FAD and FMN Flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN) are derived from riboflavin (Vit B 2 ) Flavin coenzymes are involved in oxidation-reduction reactions FMN (black), FAD (black/blue)

Thiamine Pyrophosphate (TPP) TPP is a derivative of thiamine (Vit B 1 ) TPP participates in reactions of: (1) Oxidative decarboxylation (2) Transketo- lase enzyme reactions

Pyridoxal Phosphate (PLP) PLP is derived from Vit B 6 family of vitamins PLP is a coenzyme for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, transamination)

Enzymes active sites Active site – specific region in the enzyme to which substrate molecule is bound Substrate usually is relatively small molecule Enzyme is large protein molecule Therefore substrate binds to specific area on the enzyme

Properties of Enzymes Specificity of enzymes 1.Absolute – one enzyme acts only on one substrate (example: urease decomposes only urea; arginase splits only arginine) 2.Relative – one enzyme acts on different substrates which have the same bond type (example: pepsin splits different proteins) 3.Stereospecificity – some enzymes can catalyze the transformation only substrates which are in certain geometrical configuration, cis- or trans-

Sensitivity to pH Each enzyme has maximum activity at a particular pH (optimum pH) For most enzymes the optimum pH is ~7 (there are exceptions)

-Enzyme will denature above o C -Most enzymes have temperature optimum of 37 o Each enzyme has maximum activity at a particular temperature (optimum temperature) Sensitivity to temperature

Kinetic properties of enzymes Study of the effect of substrate concentration on the rate of reaction

Leonor Michaelis and Maud Menten – first researchers who explained the shape of the rate curve (1913) During reaction enzyme molecules, E, and substrate molecules, S, combine in a reversible step to form an intermediate enzyme-substrate (ES) complex k 1, k -1, k 2, k -2 - rate constant - indicate the speed or efficiency of a reaction E + SESE + P k1k1 k2k2 k -1 k -2

- At a fixed enzyme concentration [E], the initial velocity Vo is almost linearly proportional to substrate concentration [S] when [S] is small but is nearly independent of [S] when [S] is large - Rate rises linearly as [S] increases and then levels off at high [S] (saturated) Rate of Catalysis

The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is V max [S] v o = K m + [S] The Michaelis-Menten Equation K m - Michaelis constant; V o – initial velocity caused by substrate concentration, [S]; V max – maximum velocity

Effect of enzyme concentration [E] on velocity (v) In fixed, saturating [S], the higher the concentration of enzyme, the greater the initial reaction rate This relationship will hold as long as there is enough substrate present

Enzyme inhibition In a tissue and cell different chemical agents (metabolites, substrate analogs, toxins, drugs, metal complexes etc) can inhibit the enzyme activity Inhibitor (I) binds to an enzyme and prevents the formation of ES complex or breakdown it to E + P

Reversible and irreversible inhibitors Reversible inhibitors – after combining with enzyme (EI complex is formed) can rapidly dissociate Enzyme is inactive only when bound to inhibitor EI complex is held together by weak, noncovalent interaction Three basic types of reversible inhibition: Competitive, Uncompetitive, Noncompetitive

Competitive inhibition Inhibitor has a structure similar to the substrate thus can bind to the same active site The enzyme cannot differentiate between the two compounds When inhibitor binds, prevents the substrate from binding Inhibitor can be released by increasing substrate concentration Reversible inhibition

Competitive inhibition Benzamidine competes with arginine for binding to trypsin Example of competitive inhibition

Binds to an enzyme site different from the active site Inhibitor and substrate can bind enzyme at the same time Cannot be overcome by increasing the substrate concentration Noncompetitive inhibition

Uncompetitive inhibition Uncompetitive inhibitors bind to ES not to free E This type of inhibition usually only occurs in multisubstrate reactions

Irreversible Enzyme Inhibition Irreversible inhibitors group-specific reagents substrate analogs suicide inhibitors very slow dissociation of EI complex Tightly bound through covalent or noncovalent interactions

Group-specific reagents –react with specific R groups of amino acids

Substrate analogs –structurally similar to the substrate for the enzyme -covalently modify active site residues

Inhibitor binds as a substrate and is initially processed by the normal catalytic mechanism It then generates a chemically reactive intermediate that inactivates the enzyme through covalent modification Suicide because enzyme participates in its own irreversible inhibition Suicide inhibitors

Naming of Enzymes Common names are formed by adding the suffix –ase to the name of substrate Example: - tyrosinase catalyzes oxidation of tyrosine; - cellulase catalyzes the hydrolysis of cellulose Common names don’t describe the chemistry of the reaction Trivial names Example: pepsin, catalase, trypsin. Don’t give information about the substrate, product or chemistry of the reaction

Principle of the international classification All enzymes are classified into six categories according to the type of reaction they catalyze Each enzyme has an official international name ending in –ase Each enzyme has classification number consisting of four digits: EC: First digit refers to a class of enzyme, second - to a subclass, third – to a subsubclass, and fourth means the ordinal number of enzyme in subsubclass

The Six Classes of Enzymes 1. Oxidoreductases Catalyze oxidation-reduction reactions - oxidases - peroxidases - dehydrogenases

2. Transferases Catalyze group transfer reactions

3. Hydrolases Catalyze hydrolysis reactions where water is the acceptor of the transferred group - esterases - peptidases - glycosidases

4. Lyases Catalyze lysis of a substrate, generating a double bond in a nonhydrolytic, nonoxidative elimination

5. Isomerases Catalyze isomerization reactions

6. Ligases (synthetases) Catalyze ligation, or joining of two substrates Require chemical energy (e.g. ATP)

An important first step in restoring health and well-being by helping to remedy digestive problems. Food (plant) enzymes and pancreatic (animal) enzymes are used in complementary ways to improve digestion and absorption of essential nutrients. Treatment includes enzyme supplements, coupled with healthy diet that features whole foods. Plant-derived enzymes and pancreatic enzymes can be used independently or in combination.

A chart of the numerous digestive enzymes of the body and their functions. Amylasedigests starchesBromelaina proteolytic, anti-inflammatory food enzyme from pineapple. Aids digestion of fatsCatalaseworks with SOD to reduce free radical productionCellulasedigests cellulose, the fibrous component of most vegtable matter Chymotrypsinhelps convert chyme Diastasea pontent vegtable starch digestantLactasedigests lactose, or milk sugar, (almost 65% of humans are deficient).Lipasedigests fats.Mycozymea single-celled plant enzyme for digestion of starches.Pancreatina broad spectrum, proteolytic digestive aid, derived from secretions of animal pancreas; important in degenerative disease research. Papin and chymopapainproteolytic food enzymes from unripe papaya; a veegatable pepsin for digesion of proteins. These enzymes help loosen nercotic and encrusted waste material from the intestinal walls.Pepsina proteolytic enzyme that breaks down proteins into peptides. Can digest 3500 times its weight in proteins.Proteasedigests proteinsRenninhelps digest cow's milk products.Trypsina proteoytic enzyme

enzymopathy Any of various disturbances of enzyme function, such as the genetic deficiency of a specific enzyme.

Celiakia

INBORN ERRORS OF AMINO ACIDS METABOLISM Alcaptonuria - inherited disorder of the tyrosine metabolism caused by the absence of homogentisate oxidase.  homogentisic acid is accumulated and excreted in the urine  turns a black color upon exposure to air  In children:  urine in diaper may darken  In adults:  darkening of the ear  dark spots on the on the sclera and cornea  arthritis

Maple syrup urine disease - the disorder of the oxidative decarboxylation of  -ketoacids derived from valine, isoleucine, and leucine caused by the missing or defect of branched-chain dehydrogenase. The levels of branched-chain amino acids and corresponding  -ketoacids are markedly elevated in both blood and urine. The urine has the odor of maple syrup The early symptoms:  lethargy  ketoacidosis  unrecognized disease leads to seizures, coma, and death  mental and physical retardation

Phenylketonuria is caused by an absence or deficiency of phenylalanine hydroxylase or of its tetrahydrobiopterin cofactor. Phenylalanine accumulates in all body fluids and converts to phenylpyruvate.  Defect in myelination of nerves  The brain weight is below normal.  Mental and physical retardations.  The life expectancy is drastically shortened. Diagnostic criteria:  phenylalanine level in the blood  FeCl 3 test  DNA probes (prenatal)