Overview of biochemistry

• Macromolecules are large molecules composed of thousands of covalently connected atoms • Molecular structure and function are inseparable 3... Macromolecules are polymers built from mo

Overview of Biochemistry

Instructor: Dr Nguyen Thao Trang

School of Biotechnology Semester I 2015-2016

ADVANCED BIOCHEMISTRY

Outlines

• Macromolecules

• Carbohydrates: simple and complex

• Proteins: Enzymes

• Nucleic acids: DNA

• Macromolecules are large molecules composed of thousands

of covalently connected atoms

• Molecular structure and function are inseparable

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Macromolecules are polymers built from monomers

• A polymer is a long molecule consisting of many similar

building blocks

• These small building-block molecules are called monomers

• Three of the four classes of life’s organic molecules are polymers

– Carbohydrates

– Proteins

– Nucleic acids

Macromolecules are polymers built from monomers

• Many biological molecules formed by linking a chain of monomers

• Synthesis and breakdown of polymers

– Synthesis: a dehydration reaction occurs when two monomers bond

together through the loss of a water molecule

– Breakdown: polymers are disassembled to monomers by hydrolysis, a

reaction that is essentially the reverse of the dehydration reaction

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Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Macromolecules are polymers built from monomers

• Synthesis of a polymer: dehydration reaction

Macromolecules are polymers built from monomers

• Breakdown of a polymer: hydration reaction (hydrolysis)

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Outlines

• Macromolecules

• Carbohydrates: simple and complex

• Proteins: Enzymes

• Nucleotides: ATP, NAD+

• Nucleic acids: DNA

Carbohydrates

• Carbohydrates include sugars and the polymers of sugars

• The simplest carbohydrates are monosaccharides, or single sugars

• Carbohydrate macromolecules are polysaccharides, polymers composed of many sugar building blocks

Carbohydrates: function

• Rapidly mobilized source of energy

– Monosaccharides and disaccharides

• Coupled with protein to form glycoproteins

– Important in cell membranes

Carbohydrates: simple sugars

• Monosaccharides have molecular formulas that are usually

– Glucose (C6H12O6) is the most common monosaccharide

• Monosaccharides are classified by:

– The location of the carbonyl group (as aldose or ketose)

– The number of carbons in the carbon skeleton

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Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Linear form Cyclic form

Carbohydrates: simple sugars

Carbohydrates: simple sugars

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Carbohydrates: simple sugars

Carbohydrates: simple sugars

• Cyclization of glucose and fructose:

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Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Stereochemistry of carbohydrates

Carbohydrates: disaccharides

• A disaccharide is formed when a dehydration reaction joins two monosaccharides

– This covalent bond is called a glycosidic linkage

– Synthesis of maltose from 2 glucose molecules:

– Synthesis of sucrose from glucose and fructose:

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Complex carbohydrates: polysaccharides

• Polysaccharides, the polymers of sugars, have storage and structural roles

• Structure and function of a polysaccharide are determined by its sugar monomers and the positions of glycosidic linkages

• Chitin consists of chains of glucose with N-acetyl groups

• The differences between the complex carbohydrates is in the structure – branched, unbranched, spiral, hydrogen-bonded

– Cellulose is tightly packed and hard to digest

– Starch is coiled and may be branched and is easier to digest

– Glycogen is coiled with extensive branching and is even easier to digest

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Carbohydrates: function

• Rapidly Mobilized Source of Energy

– Monosaccharides and disaccharides

• Coupled with protein to form glycoproteins

– Important in cell membranes

Storage polysaccharides

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Storage polysaccharides

• Starch: a storage polysaccharide of plants, consists entirely of

glucose monomers

– Plants store surplus starch as granules within chloroplasts

– The simplest form of starch is amylose

– Glucose molecules are linked together mainly by α-1,4 glycosidic

linkage If branched then will also have α-1,6 glycosidic linkage

– Plants used for energy storage

• Potatoes, rice, corn

– Types of starch:

• Amylose – not branched

• Amylopectin – branched, more common

Storage polysaccharides

Starch:

• Amylose:

– A linear, unbranched chain of  1,4 D glucose

– A repeating unit is a the disaccharide maltose (2 glucoses)

– Forms a coiled, relatively compact helical

structure, 6 glucoses/turn

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

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Storage polysaccharides

Starch: (the main carbohydrate source in human diet)

• Amylopectin:

– Main backbone is linear, unbranched chain of  -1,4 D glucose

– Also has branches connecting to backbone and to each other by  -1,6 linkage; branch point every 25-30 glucoses

Biochemistry, Tymoczko, Berge, Strayer

Storage polysaccharides

• Glycogen: a storage polysaccharide of animals, consists

entirely of glucose monomers

• Glucose molecules are linked mainly by α-1,4 glycosidic

linkage Glycogen is branched therefore it also has α-1,6

glycosidic linkage, branch point every 8-12 glucoses

• Found in animals: stored mainly in liver and muscle

25 Biochemistry, Tymoczko, Berge, Strayer

Carbohydrates: function

• Rapidly Mobilized Source of Energy

– Monosaccharides and disaccharides

• Coupled with protein to form glycoproteins

– Important in cell membranes

Structural polysaccharides

• Cellulose:

– Is a major component of the tough wall of plant cells

– Like starch, cellulose is a polymer of glucose, but the glycosidic linkages differ

– The difference is based on two ring forms for glucose: alpha (  ) and beta (  )

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Structural polysaccharides

• Cellulose:

– Starch: -1,4 linkage

– Cellulose: -1,4 linkage

Structural polysaccharides

• Cellulose:

– In straight structures, H atoms on one strand can bond with OH groups on other strands

– Parallel cellulose molecules held together this way are grouped into microfibrils, which form strong building

materials for plants

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Structural polysaccharides

• Cellulose:

Structural polysaccharides

• Chitin:

Outlines

• Macromolecules

• Carbohydrates: simple and complex

• Proteins: Enzymes

• Nucleotides: ATP, NAD+

• Nucleic acids: DNA

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Proteins: functions

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Proteins: functions

• There are 20 amino acids, each with

a different substitution for R

Amino acids

• At physiological pH (7.4), the amino groups are protonated

and the carboxylic acid groups are in their conjugate base

(carboxylate) form:

• Amino acids can act as both an acid and a base

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Amino acids

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Amino acids

Amino acids

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Peptide bond

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

N-terminus

C-terminus

Protein: structure and function

• A functional protein consists of one or more polypeptides precisely twisted, folded, and coiled into a unique shape

• The sequence of amino acids determines a protein’s 3D structure

• A protein’s structure determines its function

• 4 levels of protein structure:

Protein: primary structure

• The primary structure of a protein is its unique sequence of amino acids

Protein: secondary structure

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• The coils and folds of secondary structure result from hydrogen bonds between repeating constituents of the polypeptide backbone

Protein: tertiary structure

• Tertiary structure is determined by interactions between R, rather than interactions between backbone constituents in secondary structure

• These interactions between R groups include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals

interactions

• Strong covalent bonds called disulfide bridges may reinforce

the protein’s structure

Protein: tertiary structure

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Protein: quaternary structure

• Quaternary structure results when two or more polypeptide chains form one macromolecule

• Collagen is a fibrous protein consisting of three polypeptides coiled like a rope

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Collagen

Protein: quaternary structure

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• Hemoglobin is a globular protein consisting of four

Protein: structure and function

• A slight change in primary structure can affect a protein’s structure and ability to function

• Example: Sickle-cell disease, an inherited blood

disorder, results from a single amino acid substitution in the protein hemoglobin

Protein: structure and function

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Protein: what determines its structure?

• In addition to primary structure, physical and chemical conditions can affect structure

• Alterations in pH, salt concentration, temperature,

or other environmental factors can cause a protein to unfold

• This loss of a protein’s native structure is called

denaturation

• A denatured protein is biologically inactive

Protein: what determines its structure?

• In addition to primary structure, physical and chemical conditions can affect structure

• Alterations in pH, salt concentration, temperature,

or other environmental factors can cause a protein to unfold

• This loss of a protein’s native structure is called

denaturation

• A denatured protein is biologically inactive

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Proteins: Enzymes

• Enzymes are proteins that catalyze reactions: they speed up chemical reactions

• Enzymes are usually specific for their substrates

• Enzymes are not consumed (destroyed) in the process

• Some enzymes need cofactors to function

– Metals

– Organic molecules (coenzymes)

Enzymes: Catalytic mechanism

– Enzymes accelerate reactions by lowering activation energy (facilitating the formation of the transition state)

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• Enzymes provide a different pathway for the reaction through a new

lower energy pathway

• Enzymes do not affect the free energy change (G o )

Enzymes: Kinetics

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The rate of formation of P can be expressed as the product of the rate constant of the reaction yielding P and the concentration of its immediately preceding intermediate:

The overall rate of production of ES is:

Fundamentals of biochemistry-Life at the molecular level, Voet, Voet, Pratt

Enzymes: Kinetics

• The reaction velocity:

- At initial velocity (t = 0):

- When enzyme is saturated with substrate (all E is in ES form),



60 with

Michaelis-Menten equation

Enzymes: Kinetics

• Rectangular hyperpolic shape

• o approaches V max at high [S]

• 3 regions of the concentration range:

1 Very small [S]: o  [S]

2 Very large [S]: o = V max

( o is independent on [S])

3 Middle range of [S]: increase in o is

less than increase in [S]

KM = [S] when o = V max/2

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Fundamentals of biochemistry-Life at the molecular level,

Voet, Voet, Pratt

Plot of o versus [S]

Enzymes: Kinetics

– KM is the substrate concentration at which the reaction rate is half of its maximal value (KM = [S] when V o = V max/2) or when half of the active sites are filled Therefore, if an enzyme has a small value of KM, it

achieves maximal catalytic efficiency at low substrate concentrations

– KM measures the affinity of the enzyme for its substrate

If k 2 << k -1  KM = KS ( KM large: weak binding; KM small: strong binding)

– KM is unique for each enzyme–substrate pair The magnitude of KM

varies widely with the identity of the enzyme and the nature of the substrate

Enzymes: Kinetics

– Reveals the turnover number of an enzyme

– Turnover number (k 2 or k cat) is the number of substrate molecules (S) converted into products (P) in a unit time when the enzyme (E) is fully saturated with substrate (S)

[E] T is the total concentration of enzyme

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Enzymes: Kinetics

– When [S] << KM , very little ES is formed, thus [E] ~ [E]T

kcat/KM is the apparent second-order rate constant of the enzymatic

reaction; the rate of the reaction varies directly with how often enzyme and substrate encounter one another in solution

 kcat/KM is therefore a measure of an enzyme’s catalytic efficiency

- Measure enzyme’s preference for substrates

- Measure enzyme’s catalytic efficiency

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Enzymes: Kinetics

1 Preference of enzyme for different substrate (specificity):

Ex Chymotrypsin protease catalizes ester bond whose carboxylic group has bulky hydrophobic and/or aromatic groups

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Which amino acid is most preferred by chymotrypsin for contribution of

carbonyl group in ester bond to be cleaved, among substrates above?

Enzymes: Kinetics

2 Catalytic efficiency reference of enzyme for different substrate

(specificity):

- The higher k cat /K M , the more efficient the enzyme

- Maximum possible k cat /K M is dictated by the diffusion limit

Example:

1 If KM = KS, which enzyme above binds its substrate the most tightly?

2 Which enzyme has the most rapid catalytic turnover when the enzyme

is saturated with substrate?

3 Which enzymes have the highest catalytic efficiency?

Enzyme inhibitors

• Effect of inhibitors on enzyme kinetics

– Activity of many enzymes can be inhibited by the binding of specific small molecules and ions

– Substances that reduce an enzyme’s activity in this way are known as inhibitors

– Most of therapeutic drugs are enzyme inhibitors For example, AIDS is treated almost exclusively with drugs that inhibit the activities of

certain viral enzymes

– 2 types:

• Irreversible enzyme inhibitors, or inactivators

• Reversible enzyme inhibitors

Enzyme inhibitors

• Irreversible enzyme inhibitors (inactivators)

– Bind (covalently or noncovalently) to the enzyme so tightly

permanently block the enzyme’s activity – Some irreversible inhibitors are important drugs

– Examples:

• Penicillin acts by covalently modifying the enzyme transpeptidase, thereby preventing the synthesis of bacterial cell walls and thus killing the bacteria

• Aspirin acts by covalently modifying the enzyme cyclooxygenase, reducing the synthesis of signaling molecules in inflammation

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Enzyme inhibitors

• Reversible enzyme inhibitors

– Reversible enzyme inhibitors diminish an enzyme’s activity by

interacting reversibly with it

• Affects the KM

– Uncompetitive inhibitor:

• Binds only to the enzyme– substrate complex

• Cannot be overcome by the addition of more substrate

Enzyme inhibitors

• Reversible enzyme inhibitors

– Noncompetitive (or mixed) inhibitor:

• Inhibitor and substrate can bind simultaneously to an enzyme molecule at different binding sites

• Decrease the concentration of functional enzyme rather than by diminishing the amount of enzyme molecules that are bound to substrate The net effect is to decrease the turnover number (kcat) (or Vmax), KM is unaffected

• Cannot be overcome by increasing the substrate concentration

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Biochemistry, Tymoczko, Berge, Strayer

Kinetics of competitive inhibition

Here, I is the inhibitor, EI is the catalytically inactive enzyme–inhibitor complex

Assuming I binds reversibly to E, at equilibrium:

 A competitive inhibitor therefore reduces the concentration of free enzyme available for

substrate binding  K M increases Michaelis–Menten equation for a competitively inhibited reaction:

where

 is the factor by which [S] must be

increased in order to overcome the

effect of the presence of inhibitor