enzymes

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enzymes

ENZYMES AND

METABOLIC PATHWAYS

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Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


1. Chemical reactions in cells



Thousands of biochemical reactions, in which metabolites are

converted into each other and macromolecules are build up, proceed

at any given instant within living cells. However, the t

greatest

majority of these reactions would occour spontaneously at

extremely low rates.


For example, the t

oxidation of a fatty acid to carbon dioxide and water in a test

tube requires extremes of pH, high temperatures and corrosive chemicals. Yet

in the cell, , such a reaction takes place smoothly and rapidly within a narrow

range of pH and temperature. As another example, , the average protein must

be boiled for about 24 hours in a 20% HCl solution to achieve a complete

breakdown. In the body, the breakdown takes place in four hours or less under

conditions of mild physiological temperature and pH.

How can living things perform the magic of speeding up chemical

reactions many orders of magnitude, specifically those reactions

they most need at any given moment?

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


2. Introducing enzymes







The ENZYMES are the driving force behind all biochemical

reactions happening in cells.

Enzymes lower the energy barrier between reactants and products,

thus increasing the rate of the reaction.

Enzymes are biological catalysts. A catalyst is a species that

accelerates the rate of a chemical reaction whilst remaining

unchanged at the end of the reaction. Catalysis is achieved by

reducing the activation energy for the reaction.

Enzymes can catalyse reactions at rates typically 10 6 to 10 14 times

faster than the uncatalysed reaction.

Enzymes are very selective about substrates they act upon and also

where the chemistry takes place on a substrate.

Both the forward and reverse reactions are catalysed. A catalyst

cannot change the position of thermodynamic equilibrium, only the

rate at which it is attained.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


3. Enzymes are proteins






Enzymes are composed of proteins, and

proteins are long polymers of amino acids.

Amino acids all have this

general formula:

Amino acids have two functional groups (aminic and carbossilyc),

which can react together forming covalent bonds called peptide

bonds, , so that they are linked head-to-tail.

The side chain, or R group, can be anything from a hydrogen atom

(as in the amino acid glycine) ) to a complex ring (as in the amino

acid tryptophan).

Each of the 20 amino acids known to occur in proteins has a

different R group that gives it its unique properties.

The linear sequence of the amino acids in a polypeptide chain

constitutes the primary structure of the protein

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


4. Four levels of structure of the proteins

Proteins have a complex structure that is traditionally thought of as

having four levels.





The primary structure of a protein is the sequence of amino acids in its

polypeptide chain.

The secondary structure is the regular arrangement of amino acids

within localized regions of the polypeptide.

The tertiary structure is the folding of the polypeptide chain as a result

of interactions between the side chains of amino acids.

The fourth level of protein structure, quaternary structure, consists of

the interactions between different polypeptide chains in proteins

composed of more than one polypeptide.

Many proteins are compact structures; such proteins are called

globular proteins. Enzymes and antibodies are among the important

globular proteins. Other, unfolded proteins, called fibrous proteins, are

important components of such structures as hair and muscle.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


2. The sequence of aminoacids

The peptide bond. (a) A polypeptide

is formed by the removal of water

between amino acids to form peptide

bonds. Each aa indicates an amino

acid. R1, R2, and R3 represent R

groups (side chains) that

differentiate the amino acids. R can

be anything from a hydrogen atom

(as in glycine) to a complex ring (as

in tryptophan). (b) The peptide

group is a rigid planar unit with the

R groups projecting out from the CN

backbone. Standard bond distances

(in angstroms) are shown.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


5. Primary structure

Linear sequences of two proteins. (a) The E. coli tryptophan

synthetase A protein, 268 amino acids long. (b) Bovine insulin

protein. Note that the amino acid cysteine can form unique “sulfur

bridges,” because it contains sulfur.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


6. Secondary structures

The secondary structure of a protein refers to the interrelations of amino acids that are close

together in the linear sequence. Polypeptides can bend into regularly repeating (periodic)

structures, created by hydrogen bonds between the CO and NH groups of different residues.

Two of the basic periodic structures are the α helix and the β pleated sheet.

The α helix, a common basis of secondary

protein structure. Each R is a specific side

chain on one amino acid. The black dots

represent weak hydrogen bonds that bond the

CO group of residue n to the NH group of

residue n + 4, thereby stabilizing the helical

shape.

Two views of the antiparallel β pleated sheet,

another common form of secondary protein

structure. Adjacent strands run in opposite

directions. Hydrogen bonds between NH and CO

groups of adjacent strands stabilize the structure.

The side chains (R) are above and below the plane

of the sheet.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


7. Terziary structure


A protein also has a three-dimensional architecture, termed the

tertiary structure, which is created by electrostatic, hydrogen, and

Van der Waals bonds that form between the various amino acid R

groups, causing the protein chain to fold back on itself. In many

cases, amino acids that are far apart in the linear sequence are

brought close together in the tertiary structure.

Folded tertiary structure of myoglobin, an

oxygen-storage protein. Each dot represents

an amino acid. The heme group, a cofactor

that facilitates the binding of oxygen, is

shown in blue.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


8. The four levels of protein structure

Levels of protein structure.

(b) Primary structure.

(c) Secondary structure. The

polypeptide shown in part a

is drawn into an α helix by

hydrogen bonds.

(d) Tertiary structure: the three-

dimensional structure of

myoglobin.

(e) Quaternary structure: the

arrangement of two α

subunits and two β subunits

to form the complete

quaternary structure of

hemoglobin.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


9. The active site




Enzymes are typically large proteins, which are structured

specifically for the reaction they catalyze. Their size provide sites

for action and stability of the overall structure.

Two important sites within enzymes are:



The catalytic site, , which is a region within the enzyme involved with

catalysis, and

The substrate binding site which is the specific area on the enzyme to which

reactants called substrates bind to.

The catalytic site and substrate binding site are often close or

overlapping and collectively they are called the active site.


If the catalytic site is not near the substrate binding site it can move into

position once the enzyme is bound to a substrate.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


10. The “Lock-and-key” metaphor

Schematic representation

of the action of a

hypothetical enzyme in

putting two substrate

molecules together. (a) In

the "lock-and-key"

mechanism the substrates

have a complementary fit

to the enzyme's active

site. (b) In the induced-fit

model, binding of

substrates induces a

conformational change in

the enzyme.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


11. Aditional components of enzymes

Often enzymes require additional components to become

active. These may be:

co-factors: simple cations, or small organic or

inorganic molecules that bind loosely to the enzyme,

prosthetic groups: similar to co-factors but more

tightly bound to the enzyme, or

co-enzymes – which are more complex than co-factors

and prosthetic groups, they often act as a second

substrate or bind covalently with the enzyme to affect

the active site.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


12. The first step of photosynthesis

Photosynthesis. . The key passage of the photosynthesis is the organication of the

carbon, or the fixation of CO . 2

The CO 2

molecule condenses with ribulose 1,5-bisphosphate to form an unstable

six-carbon compound, which is rapidly hydrolyzed to two molecules of 3-

phosphoglycerate.

This reaction is catalyzed by the enzyme ribulose 1,5-bisphosphate carboxylase/

oxygenase (RUBISCO)

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


13. An enzyme of fundamental importance for life

The enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (RUBISCO) is located

on the stromal surface of the thylakoid membranes of chloroplasts. It comprises eight

large (L) subunits (one shown in red and the others in yellow) and eight small (S)

subunits (one shown here in blue and the others in white).

The active sites lie in the L subunits. Each L

subunit contains a catalytic site and a

regulatory site. The S chains enhance the

catalytic activity of the L chains. This enzyme

is very abundant, constituting more than 16%

of chloroplast total protein. RUBISCO is

probably the most abundant protein in the

biosphere.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


14. The active site of RUBISCO

Structure of the catalytic domain of the

active form of ribulose 1,5-bisphosphate

carboxylase.

Dark blue cylinders represent α helices

and yellow arrows represent β sheets in

the polypeptide. The key residues in the

active site are carbamylated lysine 191,

aspartate 193, and glutamate 194; a Mg 2+

ion is bound to carbamylated lysine 191.

The substrates CO 2

and ribulose 1,5-

bisphosphate are shown bound to the

active site.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


15. METABOLIC PATHWAYS




There are thousands of enzyme-catalyzed reactions in a cell. If the

biochemical reactions involved in this process were reversible, we would

convert our macromolecules back to metabolites if we stop eating even for

a short period of time.

To prevent this from happening, our metabolism is organized in metabolic

pathways. . These pathways are a series of biochemical reactions which

are, as a whole, irreversible.

These reactions are organized in consecutive steps or pathways where the

products of one reaction can become the reactants in another. Every

biochemical molecule is synthesized in a biochemical pathway with

specific enzymes.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


16. Metabolic networks

A metabolic network is the

complete set of metabolic and

physical processes that determine

the physiological and

biochemical properties of a cell.

As such, these networks

comprise the chemical reactions

of metabolism as well as the

regulatory interactions that guide

these reactions.

With the sequencing of complete

genomes, it is now possible to

reconstruct the network of

biochemical reactions in many

organisms, from bacteria to

human. Several of these

networks are available online

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


17. Metabolic pathways of phenylalanine in human

One small part of the

human metabolic

map, showing the

consequences of

various specific

enzyme failures.

(Disease phenotypes

are shown in colored

boxes.)

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


18. Defects in amino acid metabolism




The most frequent defects in amino

acid metabolism involve the amino

acids phenylalanine and tyrosine.

Numerous enzymes are required to

convert phenylalanine into a variety of

biochemical products. The metabolism

of phenylalanine and the various

metabolic blocks are illustrated in

graphic on the left where for simplicity,

many intermediate steps have been

omitted.

The circled letters indicate enzyme

defects which will of course disrupt the

reactions which follow it.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


19. Phenylketonuria

Phenylketonuria is caused by an absence or deficiency of phenylalanine hydroxylase

or, more rarely, of its tetrahydrobiopterin cofactor. . Phenylalanine accumulates in all

body fluids because it cannot be converted into tyrosine. Normally, three-quarters of

the phenylalanine is converted into tyrosine, and the other quarter becomes

incorporated into proteins. The accumulation of phenylpyruvate leads to severe

mental retardation in infants. If the high level of phenylpyruvic acid is detected soon

after birth, the baby can be placed on a special low-phenylalanine diet and develops

without retardation.

Because the major outflow pathway

is blocked in phenylketonuria, the

blood level of phenylalanine is

typically at least 20-fold as high as in

normal people. Minor fates of

phenylalanine in normal people, such

as the formation of phenylpyruvate,

become major fates in

phenylketonurics.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


20. Goitrous cretinism and Albinism

Goitrous Cretinism

Hypothyroidism results from the absence of an

enzyme to incorporate iodine into tyrosine in the first

step in the synthesis of thyroxine. The result is

stunted growth, lethargy, course hair, poor muscle

tone and other facial defects. Hypothyroidism is

treated by administration of thyroid extract.

Albinism

The biochemical defect in albinism appears to be

the absence of the enzyme tyrosinase, , which prevents

the synthesis of melanin pigment by pigment-

forming cells. These individuals have a very white

skin, fine white hair, pink or light blue irises of the

eyes, and a variety of other eye disturbances. Various

types of localized albinism are characterized by the

absence of pigment in specific parts of the body.

There is no treatment for albinism.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini


21. Tyrosinosis and Alkaptonuria

Tyrosinosis

Accumulation of p-hydroxyphenylpyruvic

acid usually leads to an enlargement of the

liver and spleen. Death results from liver

failure between 4 months and 5 years of age.

Diet control may help in reducing the

symptoms of tyrosinosis.

Alkaptonuria

Alkaptonuria occurs when the absence of an

enzyme prevents the breakdown of

homogentisic acid. A large amount of

homogentisic acid excreted in the urine causes

it to turn black upon exposure to air. Other

characteristics of alkaptonuria include arthritis

and pigmentation of cartilage. It was this

relatively benign disease that was first used as

the basis for the concept of inborn errors of

metabolism.

Genetica per Scienze Naturali

a.a. 08-09 prof S. Presciuttini

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