Regulation of serine protease activity by an engineered metal switch

A recombinant trypsin was designed whose catalytic activity can be regulated by varying the concentration of Cu2+ in solution. Substitution of Arg-96 with a His in rat trypsin (trypsin R96H) places a new imidazole group on the surface of the enzyme near the essential active-site His-57. The unique spatial orientation of these His side chains results in the formation of a stable, metal-binding site that chelates divalent first-row transition-metal ions. Occupancy of this site by a metal ion prevents the imidazole group of His-57 from participating as a general base in catalysis. As a consequence, the primary effect of the transition metal ion is to inhibit the esterase and amidase activities of trypsin R96H. The apparent Ki for this inhibition is in the micromolar range for copper, nickel, and zinc, the tightest binding being to Cu2+ at 21 microM. Trypsin R96H activity can be fully restored by removing the bound Cu2+ ion with EDTA. Multiple cycles of inhibition by Cu2+ ions and reactivation by EDTA demonstrate that reversible regulatory control has been introduced into the enzyme. These results describe a novel mode of inhibition of serine protease activity that may also prove applicable to other proteins.