ENZYMES: CLASSIFICATION, STRUCTUREENZYMES: CLASSIFICATION, STRUCTURE

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 enzymesEnzymes

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

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-adenosylmethioninedonates methyl groups in many biosynthesis reactions

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 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

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

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 EnzymesSpecificity 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 pHEach 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 45-50oC

-Most enzymes have temperature optimum of 37o

Each enzyme has maximum activity at a particular temperature (optimum temperature)

Sensitivity to temperature

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 celluloseCommon names don’t describe the chemistry of the reaction Trivial namesExample: 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)

ENZYMES: KINETICS, ENZYMES: KINETICS, INHIBITION, REGULATIONINHIBITION, REGULATION

Kinetic properties of enzymesStudy of the effect of substrate concentration on the rate of reaction

- 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

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

k1, k-1, k2, k-2 - rate constant - indicate the speed or efficiency of a reaction

E + S ES E + P k1 k2

k-1 k-2

The basic equation derived by Michaelis and Menten to explain enzyme-catalyzed reactions is

Vmax[S]

vo =

Km + [S]

The Michaelis-Menten Equation

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

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

Regulation of enzyme activity

• Allosteric control• Reversible covalent modification• Isozymes (isoenzymes)• Proteolytic activation

Methods of regulation of enzyme activity

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

Allosteric enzymes

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

• 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

Example of allosteric enzyme - phosphofructokinase-1 (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 phospho-rylation are called protein kinases

Enzymes taking part in dephosphorylation are called phosphatases

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

Isoenzymes (isozymes)

Some metabolic processes are regulated by enzymes that exist in different molecular forms - isoenzymes

• H4: highest affinity; best in aerobic environment•M4: lowest affinity; best in anaerobic environment

Isoenzymes are important for diagnosis of different diseases

There are 5 Isozymes of LDG: H4 – heart HM3

H2M2

H3M M4 – liver, muscle

Lactate dehydrogenase – tetramer (four subunits) composed of two types of polypeptide chains, M and H

Example: lactate dehydrogenase (LDG) Lactate + NAD+ pyruvate + NADH + H+

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 - 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

Multienzyme Complexes and Multifunctional Enzymes

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