EFFECT OF LYS175 MUTATION ON STRUCTURE-FUNCTION PROPERTIES OF PROPIONIBACTERIUM-SHERMANII SUPEROXIDE-DISMUTASE

The role of electrostatic factors in the enzyme-substrate encounter pr ocess of manganese and iron containing superoxide dismutases has been studied in the enzyme from Propionibacterium shermanii by chemical neu tralization of lysine residues and site-directed mutagenesis of the hi ghly conserved residue Lys175. Lysine residues have been neutralized b y carbamoylation and Lys175 has been selectively replaced by isoleucin e and arginine, Catalytic measurements show a dramatic decrease of the activity in the chemically modified enzyme, Electrostatic potential c alculations evidence in the modified enzyme a large contraction of the positive potential areas which surround the active sites in the-nativ e enzyme, indicating that electrostatic factors are critical in the en zyme-substrate encounter process of Mn- and Fe-superoxide dismutases. The activity drastically decreases also in Lys175-->Ile but not in the Lys175-->Arg mutant. Brownian dynamics simulations indicate that the decrease of activity in the Lys175-->Ile mutant cannot be due only to a decrease of the enzyme-substrate association rate, suggesting that L ys175 plays a relevant role also in the structural stabilization of th e active site.