Introduction. Structure, properties and biological functions of proteins. Methods of secretion and purification. Peptides. Complex proteins, their biological role. Structure and physical-chemical properties of enzymes

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

Coenzyme classification 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 Vitamin-derived coenzymes - derivatives of vitamins Vitamins cannot be synthesized by mammals, but must be obtained as nutrients

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

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

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

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

FAD and FMN Flavin adenine dinucleotide (FAD) and Flavin mononucleotide (FMN) are derived from riboflavin (Vit B2) Flavin coenzymes are involved in oxidation-reduction reactions FMN (black), FAD (black/blue)

Thiamine Pyrophosphate (TPP) TPP is a derivative of thiamine (Vit B1) TPP participates in reactions of: (1) Oxidative decarboxylation (2) Transketo-lase enzyme reactions

Pyridoxal Phosphate (PLP) PLP is derived from Vit B6 family of vitamins PLP is a coenzyme for enzymes catalyzing reactions involving amino acid metabolism (isomerizations, decarboxylations, transamination)

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

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

Active site contains functional groups (-OH, -NH, -COO etc) Binds substrates through multiple weak interactions (noncovalent bonds)

Theories of active site-substrate interaction Fischer theory (lock and key model) The enzyme active site (lock) is able to accept only a specific type of substrate (key)

Koshland theory (induced-fit model) The process of substrate binding induces specific conformational changes in the the active site region

Specificity of enzymes Properties of Enzymes Specificity of enzymes Absolute – one enzyme acts only on one substrate (example: urease decomposes only urea; arginase splits only arginine) Relative – one enzyme acts on different substrates which have the same bond type (example: pepsin splits different proteins) 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)

Sensitivity to temperature Each enzyme has maximum activity at a particular temperature (optimum temperature) -Enzyme will denature above 45-50oC -Most enzymes have temperature optimum of 37o

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

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)

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

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

Reversible inhibition 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

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

Noncompetitive 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

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

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

Suicide inhibitors •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

Regulation of enzyme activity Methods of regulation of enzyme activity • Allosteric control • Reversible covalent modification • Isozymes (isoenzymes) • Proteolytic activation

Allosteric enzymes Allosteric enzymes have a second regulatory site (allosteric site) distinct from the active site Allosteric enzymes contain more than one polypeptide chain (have quaternary structure). Allosteric modulators bind noncovalently to allosteric site and regulate enzyme activity via conformational changes

Example of allosteric enzyme - phosphofructokinase-1 (PFK-1) PFK-1 catalyzes an early step in glycolysis Phosphoenol pyruvate (PEP), an intermediate near the end of the pathway is an allosteric inhibitor of PFK-1 PEP

Phosphorylation reaction

Dephosphorylation reaction Usually phosphorylated enzymes are active, but there are exceptions (glycogen synthase) Enzymes taking part in phospho-rylation are called protein kinases Enzymes taking part in dephosphorylation are called phosphatases

Isoenzymes (isozymes) Some metabolic processes are regulated by enzymes that exist in different molecular forms - isoenzymes Isoenzymes - multiple forms of an enzyme which differ in amino acid sequence but catalyze the same reaction Isoenzymes can differ in: kinetics, regulatory properties, the form of coenzyme they prefer and distribution in cell and tissues Isoenzymes are coded by different genes

Example: lactate dehydrogenase (LDG) Lactate + NAD+ pyruvate + NADH + H+ Lactate dehydrogenase – tetramer (four subunits) composed of two types of polypeptide chains, M and H There are 5 Isozymes of LDG: H4 – heart HM3 H2M2 H3M M4 – liver, muscle • H4: highest affinity; best in aerobic environment •M4: lowest affinity; best in anaerobic environment Isoenzymes are important for diagnosis of different diseases

Examples of specific proteolysis Activation by proteolytic cleavage • Many enzymes are synthesized as inactive precursors (zymogens) that are activated by proteolytic cleavage • Proteolytic activation only occurs once in the life of an enzyme molecule Examples of specific proteolysis •Digestive enzymes –Synthesized as zymogens in stomach and pancreas •Blood clotting enzymes –Cascade of proteolytic activations •Protein hormones –Proinsulin to insulin by removal of a peptide