Metal-ion catalysis Metals can function catalytically in several ways

1. Metal-ion catalysis Metals can function catalytically in several ways * May serve as an electrophilic catalyst, stabilising a negative charge on a reaction intermediate * May generate a nucleophile by increasing the acidity of a nearby molecule  carbonic anhydrase *May bind to substrate, increasing the binding energy  adenylate kinase

2. Carbonic anhydrase Important enzyme in humans for a variety of transport processes * 7 human genes, substantial sequence identity * Mutations cause osteopetrosis (excessive formation of dense bones accompanied by anemia) and mental retardation * Extremely fast enzyme: kcat = 106 s-1

10. Proton shuttle The catalytic function of carbonic anhydrase has been enhanced through the evolution of an apparatus for controlling proton transfer from and to the active site * His64 abstracts proton from the zinc bound water * The buffer removes a proton from His64

12. Convergent evolution Carbonic anhydrases from animals, bacteria, algae and plants * -carbonic anhydrase animals, bacteria, algae 3 His conserved * -carbonic anhydrase bacteria, plants 1 His conserved, 2 Cys * -carbonic anhydrase archeon 3 Zn binding sites

14. Restriction endonucleases Perform highly specific DNA-cleavage reactions * Cleave DNA at specific recognition sequences * Cleave cognate DNA molecules one million-fold as efficiently as nonspecific sites * Must not degrade the host DNA

16. EcoRV endonuclease Within the enzyme-substrate complex, the DNA substrate is distorted in a manner that generates a magnesium ion binding site between the enzyme and DNA The Mg2+ ion binds and activates a water molecule, which attacks the phosphodiester backbone EcoRV may bind non-specific DNA molecules, but these molecules are not distorted in a manner that allows Mg2+ binding, and hence, catalysis

19. EcoRV endonuclease specificity Recognition sequences are inverted repeats Recognition site has two-fold rotational symmetry Restriction enzyme displays a corresponding symmetry: homodimer with symmetric subunits Enzyme surrounds the DNA in a tight embrace

26. Evolutionary history type II restriction enzymes No significant sequence similarity * Conserved catalytic core * Bacteria may have obtained genes encoding these enzymes from other species by horizontal gene transfer * The passing between species of pieces of DNA (such as plasmids) that provide a selective advantage in a particular environment