ENZYMES: CLASSIFICATION, STRUCTURE
Enzymes - catalysts of biological reactions Accelerate reactions by a millions fold
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
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) Apoenzyme (protein part) Simple (only protein) Cofactor Prosthetic groups Coenzyme -usually small inorganic molecule or atom; -large organic molecule -usually tightly bound to apoenzyme -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
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 can donate phosphoryl group ATP S-adenosylmethionine donates methyl groups in many biosynthesis reactions S-adenosylmethionine
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 dehydrogenases
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) Transketolase 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 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
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)
Sensitivity to temperature Each enzyme has maximum activity at a particular temperature (optimum temperature) -Enzyme will denature above 4550oC -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 Trivial names reaction 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 -
2. Transferases Catalyze group transfer reactions
3. Hydrolases Catalyze hydrolysis reactions where water is the acceptor of the transferred group - esterases peptidases -
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)
ENZYMES: KINETICS, INHIBITION, REGULATION
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)
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 E + S k1 k-1 ES k2 E + P k-2 k1, k-1, k2, k-2 - rate constant - indicate the speed or efficiency of a reaction
The Michaelis-Menten Equation The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is Vmax[S] vo = Km + [S] 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
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
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
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
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
Regulation of enzyme activity by covalent modification Covalent attachment of a molecule to an amino acid side chain of a protein can modify activity of enzyme
Phosphorylation reaction
Dephosphorylation reaction Usually phosphorylated enzymes are active, but there are exceptions (glycogen synthase) Enzymes taking part in phosphorylation 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 H 2M 2 H 3M 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
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
Multienzyme Complexes and Multifunctional Enzymes Multienzyme complexes - different enzymes that catalyze sequential reactions in the same pathway are bound together Multifunctional enzymes - different activities may be found on a single, multifunctional polypeptide chain
Metabolite channeling Metabolite channeling - channeling of reactants between active sites Occurs when the product of one reaction is transferred directly to the next active site without entering the bulk solvent Can greatly increase rate of a reactions Channeling is possible in multienzyme complexes and multifunctional enzymes