MOLECULAR BIOLOGY (PART II) Flashcards
ENZYMES
Enzymes are complex, three dimensional, globular proteins. They act as catalysts in biological reactions (biocatalysts), that is, they speed up the rate of the reactions.
Each enzyme has a specific molecule where it acts and this is called the SUBSTRATE. Usually the substrate is smaller than the enzyme and therefore only a small part of the enzyme comes into contact with the substrate. This region of the enzyme is called the ACTIVE SITE of the enzyme.
Only a few amino acids of the enzyme molecule make up the active site. These amino acids are often brought together by the folding of the chain.
MECHANISM OF ENZYME ACTION: THE KEY-LOCK MECHANISM
Enzymes show high specificity for their substrates. The substrate matches the active site and is chemically attracted to it. The substrate binds to the active site since collisions are made between the two molecules found in a liquid state. In the same way a key fits a lock very precisely, so the substrate (key) fits accurately into the active site (lock). The products of the reaction have a different shape than the substrate and so, as soon as they are formed, they escape from the active site, leaving it free to become attached to another substrate molecule.
Enzyme properties
- High specificity for the substrate
- Act in small concentrations
- Remain unchanged after the end of the reaction
Factors affecting enzyme activity OR factors affecting the rate of an enzyme reaction
The rate of an enzyme reaction is measured as the amount of products formed (or the amount of substrate used) in a certain period of time. There are 4 factors which affect the rate of an enzyme reaction.
- Substrate concentration
For a given enzyme concentration the rate of an enzyme reaction will increase proportionally upon increase of the substrate concentration.
This happens up to a point. At this point all active sites of enzymes are used by substrates, so a further increase of the substrate concentration cannot increase the rate of the reaction, which will remain constant, independent of the substrate concentration. The rate reaches a plateau.
- Temperature
As temperature increases kinetic energy of both substrate and enzyme increases-they move faster, the rate of reaction increases. There is an optimum temperature at which each enzyme works best. Usually this is limited between 250 and 400C. In very high temperatures the enzymes are denatured, the three-dimensional structure is destroyed, the enzyme stops functioning, the rate of the reaction drops.
- pH
Each enzyme has an optimum pH at which it works best-the rate is highest. Lower or higher pH values will lower the enzyme activity or stop it completely because the enzyme gets denatured. Both acids and alkalis may denature enzymes.
Immobilised enzymes are used in industry
Enzymes are widely used in industry for various functions (to produce detergents with enzymes, to produce soft drinks, to cure food such as meat, ham etc). These enzymes are used as immobilized, that is , attachment of enzymes into another material which restricts their movement. Benefits of enzyme immobilization are:
• Enzymes can be reused, less costly procedure
• The products do not contain the enzyme
• Enzyme concentration can be controlled easier
There are various methods of producing immobilized enzymes:
• Attaching them onto surfaces such as glass (adsorption)
• Trapping them on a membrane or a gel (alginates)
• Creating aggregations of enzymes with a diameter οf up to 0.1mm
Use of Lactase to produce lactose-free milk
Lactose is the milk sugar.
lactose lactase glucose + galactose
sweeter than lactose
Lactase is the enzyme of the reaction and is extracted and purified by a yeast species
Kluveromyces lactis (which naturally exists in milk). Lactase is used in industries to:
• Obtain lactose-free milk needed by some people who are intolerant to lactose
• Make milk sweeter-no extra sugar is needed to be added
• Manufacture ice-cream. Lactose gives a crystalline texture to ice-creams, not glucose
and galactose
• Make yogurt easier and quicker since glucose and galactose are fermented quicker than
lactose by bacteria turning milk into yogurt The use of lactase is done by two ways:
• It is added to milk and therefore the final product contains the enzyme
• It is used as immobilized on a surface or on beads (alginates) of a porous material. Milk
allows to flow through this material. The final product does not contain lactase.
DNA REPLICATION
Through this process one DNA molecule copies itself and therefore two exact copies are formed. The process occurs as following:
A helical portion of DNA unwinds and the two strands break open between the hydrogen bonds, found between complementary bases. This process is done by the
action of an enzyme called DNA helicase.
Then another enzyme, DNA polymerase, comes and adds nucleotides to their complementary bases. Opposite to an Adenine a Thymine is added and opposite to a Guanine a Cytosine is added and the vice versa. By this way the base sequence of DNA is conserved into the new synthesized molecule.
One by one the nucleotides are linked together to form a complementary strand. This continues until the entire DNA molecule has been replicated. This type of replication is called semi-conservative because it leads to the production of two molecules; each one has one old and one new strand.
DNA TRANSCRIPTION
Protein synthesis, in order to take place, needs another intermediate molecule to carry the information from the DNA to the cytoplasm to get translated. This molecule is the mRNA. It is synthesized in the nucleus along with the other types of RNA and then exits the nucleus and acts in the cytoplasm.
The DNA double helix unwinds and only one strand is used as a template for RNA
polymerase to add nucleotides in a complementary way. The strand of DNA used as a template is called the antisense strand, while the other which is not transcribed but it is identical to the mRNA formed, is called the sense strand. When the entire part of the DNA is transcribed, mRNA exits the nucleus from the nuclear pores and moves to the cytoplasm for translation.
The genetic code
How can the genetic instructions stored in the DNA be used for the construction of new cells and organisms?
In most organisms all the chemicals are the same (carbohydrates, lipids). Only DNA and proteins differ from organism to organism. It is reasonable, therefore, to try to
understand how these two molecules correlate and how the DNA provides a “code” to synthesise proteins.
It should be reminded here that enzymes are responsible for all reactions which occur within the body and that enzymes are proteins with one polypeptide chain.
The sequence of bases of the DNA molecule makes the different varieties of DNA. This sequence is important to give the correct sequence of amino acids on the polypeptide chain.
A code is used here to correlate these two different sequences. The code is a triplet of
bases (three bases) corresponding to one amino acid.
This combination will give 64 (43) different triplets which will code for the 20 amino acids. In fact there are more triplets to code for only one amino acid.
Each triplet on the mRNA is called a codon. The genetic code is therefore a table which includes all 64 codons and their corresponding amino acids.
Properties of the genetic code:
1) It is universal- The same code is found from bacteria to humans. It declares the common origin of all species.
2) It is degenerate – more than one triplets (codons) code for the same amino acid.
There are also three triplets (UAG, UGA, UAA) that are called stop codons and do not code for any amino acid. Protein synthesis ends when such a triplet is found of the mRNA. One triplet (AUG-met) is the start triplet for translation.
TRANSLATION (PROTEIN SYNTHESIS)
translation is the process where a specific sequence of amino acids is formed in accordance to the codons found on the mRNA. Substances needed for translation: • Enzymes • Energy in the form of ATP • mRNA • ribosomes • tRNA tRNA is the kind of RNA which carries the amino acids on the translating mRNA. Each tRNA contains in one end a triplet of bases called the anticodon and is complementary to each codon triplet found on the mRNA. On the other end it carries one amino acid which comes from the corresponding codon in the genetic code.
PROCESS OF TRANSLATION
The process of translation occurs as following:
A group of ribosomes becomes attached to the mRNA for translation. The complementary anticodon of a tRNA-amino acid is attached to the first codon on the mRNA. A second codon likewise attracts its complementary anticodon tRNA-amino acid. The ribosome acts as a framework holding the mRNA and tRNAs-amino acids together until the two amino acids form a peptide bond between each other. The first tRNA is released from the ribosome and the dipeptide is carried by the second amino acid. Then the ribosome will move along the mRNA to hold the next codon-tRNA-anticodon complex together, until the third amino acid is linked with the second. By this way a polypeptide is assembled, by adding one amino acid at a time. Translation will end when the ribosome meets a stop codon (codes for no amino acid) and the polypeptide chain will be released from the complex ribosome-mRNA.
