Common features for enzymes and inorganic catalysts:

Biochemistry - as science. Structure and properties of enzymes. The mechanism of enzymes activity. Isoenzymes. Classification of enzymes. Basic principles of metabolism. Common pathways of proteins, carbohydrates and lipids transformation.

Common features for enzymes and inorganic catalysts: 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

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

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

Characteristics of active sites • Specificity(absolute, relative (group), stereospecificity) • Small three dimensional region of the protein. Substrate interacts with only three to five amino acid residues. Residues can be far apart in sequence • Binds substrates through multiple weak interactions (noncovalent bonds) • There are contact and catalytic regions in the active site

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)

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 particularpH (optimum pH) For most enzymes the optimum pH is ~7 (there are exceptions)

Sensitivity to temperature Each enzyme has maximum activity at a particulartemperature (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

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 hasclassification number consisting of four digits: EC: 2.3.4.2 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)

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

Rate of Catalysis • 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)

Vmax[S] vo = Km + [S] The Michaelis-Menten Equation The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is Km- Michaelis constant; Vo – initial velocity caused by substrate concentration, [S]; Vmax – 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

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

Competitive inhibition 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 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

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 modulatorsbind noncovalently to allosteric site and regulate enzyme activity via conformational changes

2 types of modulators(inhibitors or activators) • • Negative modulator (inhibitor) • –binds to the allosteric site and inhibits the action of the enzyme • –usually it is the end product of a biosynthetic pathway - end-product (feedback) inhibition • • Positive modulator (activator) • –binds to the allosteric site and stimulates activity • –usually it is the substrate of the reaction

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

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 (LDH) 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 LDH: • 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

Feedback inhibition • Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway) Feed-forward activation • Metabolite early in the pathway activates an enzyme further down the pathway

Stages of metabolism Catabolism Stage I. Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks. Stage II. Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl CoA) Stage III. Acetyl CoA is oxidized in citric acid cycle to CO2 and water. As result reduced cofactor, NADH2 and FADH2, are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis.

Glycerol Catabolism

Common features for enzymes and inorganic catalysts: