Structural Biochemistry/Enzyme Catalytic Mechanism/HindIII

HindIII

HindIII is a type II restriction enzyme derived from Haemophilus Influenzae. HindIII is site specific and cleaves the DNA sequence AAGCTT through hydrolysis when the cofactor Mg2+ is present. The cleavage of this sequence results in 5' sticky ends:

5'-A |A G C T T-3'

3'-T T C G A| A-5'

This type II restriction enzyme is composed of four β-sheets and one α-helix. As a type II restriction enzyme, HindIII protects the host genome against foreign DNA.

Proposed Mechanism and Usage

HindIII first binds to the DNA backbone through hydrogen bonds and weaker forces such as Van der Waals. When arrived at the specific sequence, it was suggested that HindIII hydrolyses the DNA by having the Lys-92 stabilizes the nucleophilic water while the leaving hydroxide anion is stabilized by the Asp90.

As a type II restriction protein, HindIII is useful in that it cleaves DNA at very specific sites, different than those of type 1, which cleaves randomly at sites other than the specific site. As such, HindIII is used for genetic engineering and molecular biology. It becomes possible to add, delete, or change specific genes, which is very important when trying to change an organism's genome.

It is a type II site-specific deoxyribonuclease restriction enzyme isolated from Haemophilus influenzae that cleaves the palindromic DNA sequence AAGCTT in the presence of the cofactor Mg2+ via hydrolysis. The cleavage of this sequence between the AA's results in 5' overhangs on the DNA called sticky ends.

References

Tang, D et al. (2000). "Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity". Protein Engineering Horton, N., Newberry, K. Perona, J. (1999). "Metal ion-mediated substrate-assisted catalysis in type II restriction endonucleases". Proc Natl Acad Sci U S A.