MUTAGENESIS OF RAT-LIVER ARGINASE EXPRESSED IN ESCHERICHIA-COLI - ROLE OF CONSERVED HISTIDINES

Rat liver arginase has been overexpressed in Escherichia coil using a T7-based expression system. The kinetic properties of the recombinant wild-type protein are essentially identical to those of the native rat liver enzyme. The recombinant wild-type protein contains six Mn(II) i ons per trimer, in good agreement with results obtained with the fully active native enzyme. However, in contrast to the native enzyme which loses three Mn(II) per trimer upon extended dialysis, the recombinant protein binds Mn(II) tenaciously, and retains six Mn(II) per trimer e ven after extensive dialysis. Three histidine residues, corresponding to His101, His126, and His141 in the rat liver enzyme, are highly cons erved in arginases from evolutionarily divergent species. The replacem ent of His101 and His126 with Asn by site-directed mutagenesis produce d only modest effects on enzymatic activity when measured in the prese nce of Mn(II) ions. However, EDTA treatment of these mutant enzymes re duced activity to <0.2% of that for the wild-type enzyme. The activity of wild-type enzyme and the His141Asn mutant was unaffected by treatm ent with EDTA. Thus, His101 and His126 are proposed to be ligands to t he binuclear Mn(II) center of the enzyme. The His141Asn mutation produ ced an enzyme which, in contrast to the native, wild-type, His101Asn, and His126Asn arginases, was not inactivated by diethyl pyrocarbonate. These results suggest a catalytic role for His141.