Enzymes: Biological Catalysts and Molecular Scissors, Study notes of Biochemistry

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Enzymes

Molecular Biology

Enzymes

• Enzymes are proteins
  • biological catalysts  help drive biochemical reactions
• Enzyme names end with an ase (eg., endonuclease)

It’s shape that matters!

• Lock & Key model
  • shape of protein allows enzyme & substrate to fit
  • specific enzyme for each specific reaction

Nomenclature

• Smith and Nathans (1973) proposed enzyme naming scheme - three-letter acronym for each enzyme derived from the source organism - First letter from genus - Next two letters represent species - Additional letter or number represent the strain or serotypes
• For example. the enzyme Hind II was isolated from Haemophilus influenzae serotype d.

Picking a palindrome

Words that read the same forwards as backwards

Hannah

Level

Madam

hannaH

leveL

madaM

Palindromes in DNA sequences

Genetic palindromes are similar to verbal palindromes. A palindromic sequence in DNA is one in which the 5’ to 3’ base pair sequence is identical on both strands (the 5’ and 3’ ends refers to the chemical structure of the DNA).

Discovery of Restriction Enzymes

• Arbor and Dussoix in 1962 discovered that certain bacteria contain Endonucleases which have the ability to cleave DNA.
• In 1970 Smith and colleagues purified and characterized the cleavage site of a Restriction Enzyme.
• Werner Arbor, Hamilton Smith and Daniel Nathans shared the 1978 Nobel prize for Medicine and Physiology for their discovery of Restriction Enzymes.

Restriction enzymes recognize and make a cut

within specific palindromic sequences,

known as restriction sites , in the genetic

code. This is usually a 4- or 6 base pair

sequence.

Example?

Once the recognition site was found

Ha e III could go to work cutting

(cleaving) the DNA

5’ TGACGGGTTCGA GGCC AG 3’

3’ ACTGCCCAAGGT CCGG TC 5’

blunt end

sticky end

“blunt ends” and “sticky ends”

Remember how Hae III produced a “blunt end”?

Eco RI, for instance, makes a staggered cut and produces a “sticky end” 5’ GAATTC 3’ 3’ CTTAAG 5’

5’ GA ATTC 3’ 3’ CTTA AG 5’

5’ G AATTC 3’ 3’ CTTAA G 5’

Some more examples of restriction sites of

restriction enzymes with their cut sites:

Hind III: 5’ AAGCTT 3’ 3’ TTCGAA 5’

Bam HI: 5’ GGATCC 3’ 3’ CCTAGG 5’

Alu I: 5’ AGCT 3’ 3’ TCGA 5’

Restriction Endonucleases

• Restriction endonucleases RESTRICT viruses
  • Viral genome is destroyed upon entry
• Restriction endonuclease = Restriction enzymes
  • Endo (inside), nuclease (cuts nucleic acid)
• Restriction endonuclease recognizes a short and specific DNA sequence and cuts it from inside.
• The specific DNA sequence is called recognition sequence

Few Restriction Enzymes

Enzyme Organism from which derived

Target sequence (cut at ) 5' -->3' Bam HI Bacillus amyloliquefaciens G G A T C C Eco RI Escherichia coli RY 13 G* A A T T C

Hind III Haemophilus inflenzae Rd A* A G C T T Mbo I Moraxella bovis *G A T C Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G Taq I Thermophilus aquaticus T * C G A Xma I Xanthamonas malvacearum C * C C G G G