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.

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:

Structure of enzymesEnzyme

sComplex 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

Specific features of enzymes:1. Accelerate reactions in much higher degree than inorganic catalysts

2. Specificity of action3. Sensitivity to temperature

4. Sensitivity to pH

Metalloenzymes contain firmly bound metal ions at the enzyme active sites (examples: iron, zinc,

copper, cobalt).

Example of metalloenzyme: carbonic anhydrase contains

zinc

Example of prosthetic group

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

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 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)

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 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

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 EnzymesCommon 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: 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 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

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

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

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

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 LDH: 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 (LDH) Lactate + NAD+ pyruvate + NADH + H+

• 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)

Feedback inhibition

• Metabolite early in the pathway activates an enzyme further down the pathway

Feed-forward activation

Stages of metabolismCatabolismStage 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

Catabolism is characterized by convergence of three major routs toward a final common pathway.

Different proteins, fats and carbohydrates enter the same pathway – tricarboxylic acid cycle.

Anabolism can also be divided into stages, however the anabolic pathways are characterized by divergence.

Monosaccharide synthesis begin with CO2, oxaloacetate, pyruvate or lactate. Amino acids are synthesized from acetyl CoA, pyruvate or keto acids of Krebs cycle. Fatty acids are constructed from acetyl CoA.

On the next stage monosaccharides, amino acids and fatty acids are used for the synthesis of polysaccharides, proteins and fats.

•Compartmentation of metabolic processes permits:

- separate pools of metabolites within a cell

- simultaneous operation of opposing metabolic paths

- high local concentrations of metabolites

•Example: fatty acid synthesis enzymes (cytosol), fatty acid breakdown enzymes (mitochondria)

Compartmentation of Metabolic Processes in Cell

Compartmentation of metabolic processes

Pyruvate formed in the aerobic conditions undergoes conversion to acetyl CoA by pyruvate dehydrogenase complex.

Pyruvate dehydrogenase complex is a bridge between glycolysis and aerobic metabolism – citric acid cycle.

Pyruvate dehydrogenase complex and enzymes of cytric acid cycle are located in the matrix of mitochondria.

OXIDATIVE DECARBOXYLATION OF OXIDATIVE DECARBOXYLATION OF PYRUVATEPYRUVATE

Pyruvate translocase, protein embedded into the inner membrane, transports pyruvate from the intermembrane space into the matrix in symport with H+ and exchange (antiport) for OH-.

Entry of Pyruvate into the MitochondrionPyruvate freely diffuses through the outer membrane of

mitochon-dria through the channels formed by transmembrane proteins porins.

•Pyruvate dehydrogenase complex (PDH complex) is a multienzyme complex containing 3 enzymes, 5 coenzymes and other proteins.

Conversion of Pyruvate to Acetyl CoA

Pyruvate dehydrogenase complex is giant, with molecular mass ranging from 4 to 10 million daltons.

Electron micrograph of the pyruvate

dehydrogenase complex from E. coli.

Enzymes:E1 = pyruvate dehydrogenaseE2 = dihydrolipoyl acetyltransferaseE3 = dihydrolipoyl dehydrogenase

Coenzymes: TPP (thiamine pyrophosphate), lipoamide, HS-CoA, FAD+, NAD+.

TPP is a prosthetic group of E1; lipoamide is a prosthetic group of E2; and FAD is a prosthetic group of E3. The building block of TPP is vitamin B1 (thiamin); NAD – vitamin B5 (nicotinamide); FAD – vitamin B2 (riboflavin), HS-CoA – vitamin B3 (pantothenic acid), lipoamide – lipoic acid

Overall reaction of pyruvate dehydrogenase complex

Pyruvate dehydrogenase complex is a classic example of multienzyme complex

The oxidative decarboxylation of pyruvate catalized by pyruvate dehydrogenase complex occurs in five steps.

Glucose

Glucose-6-phosphate

Pyruvate

Glycogen Ribose, NADPH

Pentose phosphate pathway

Synthesis of glycogen

Degradation of glycogen

Glycolysis Gluconeogenesis

LactateEthanol

Acetyl Co AFatty Acids Amino Acids

The citric acid cycle is the final common pathway for the oxidation of fuel molecules — amino acids, fatty acids, and carbohydrates.

Most fuel molecules enter the

cycle as acetyl

coenzyme A.

Names:

The Citric Acid Cycle

Tricarboxylic Acid Cycle Krebs Cycle

In eukaryotes the reactions of the citric acid cycle take place inside mitochondria

Hans Adolf Krebs. Biochemist; born in Germany. Worked in Britain. His discovery in 1937 of the ‘Krebs cycle’ of chemical reactions was critical to the understanding of cell metabolism and earned him the 1953 Nobel Prize for Physiology or Medicine.Physiology or Medicine.

An Overview of the Citric Acid Cycle A four-carbon oxaloacetate condenses with a two-carbon acetyl unit to yield a six-carbon citrate.

An isomer of citrate is oxidatively decarboxylated and five-carbon -ketoglutarate is formed.

-ketoglutarate is oxidatively decarboxylated to yield a four-carbon succinate.

Oxaloacetate is then regenerated from succinate.

Two carbon atoms (acetyl CoA) enter the cycle and two carbon atoms leave the cycle in the form of two molecules of carbon dioxide.

Three hydride ions (six electrons) are transferred to three molecules of NAD+, one pair of hydrogen atoms (two electrons) is transferred to one molecule of FAD.

The function of the citric acid cycle is the harvesting of high-energy electrons from acetyl CoA.

1. Citrate Synthase•Citrate formed from acetyl CoA and oxaloacetate

•Only cycle reaction with C-C bond formation

•Addition of C2 unit (acetyl) to the keto double bond of C4 acid, oxaloacetate, to produce C6 compound, citrate

citrate synthase

2. Aconitase

•Elimination of H2O from citrate to form C=C bond of cis-aconitate

•Stereospecific addition of H2O to cis-aconitate to form isocitrate

aconitase aconitase

3. Isocitrate Dehydrogenase• Oxidative decarboxylation of isocitrate to

a-ketoglutarate (a metabolically irreversible reaction)

• One of four oxidation-reduction reactions of the cycle

• Hydride ion from the C-2 of isocitrate is transferred to NAD+ to form NADH

• Oxalosuccinate is decarboxylated to a-ketoglutarate

isocitrate dehydrogenase isocitrate dehydrogenase

4. The -Ketoglutarate Dehydrogenase Complex

•Similar to pyruvate dehydrogenase complex•Same coenzymes, identical mechanisms

E1 - a-ketoglutarate dehydrogenase (with TPP) E2 – dihydrolipoyl succinyltransferase (with flexible lipoamide prosthetic group) E3 - dihydrolipoyl dehydrogenase (with FAD)

-ketoglutarate dehydrogenase

5. Succinyl-CoA Synthetase•Free energy in thioester bond of succinyl CoA is

conserved as GTP or ATP in higher animals (or ATP in plants, some bacteria)

•Substrate level phosphorylation reaction

HS-+

GTP + ADP GDP + ATP

Succinyl-CoA Synthetase

• Complex of several polypeptides, an FAD prosthetic group and iron-sulfur clusters

• Embedded in the inner mitochondrial membrane

• Electrons are transferred from succinate to FAD and then to ubiquinone (Q) in electron transport chain

• Dehydrogenation is stereospecific; only the trans isomer is formed

6. The Succinate Dehydrogenase Complex

Succinate Dehydrogenase

7. Fumarase

•Stereospecific trans addition of water to the double bond of fumarate to form L-malate

•Only the L isomer of malate is formed

Fumarase

8. Malate Dehydrogenase

Malate Dehydrogenase

Malate is oxidized to form oxaloacetate.