CONTRIBUTION OF LONG-RANGE ELECTROSTATIC INTERACTIONS TO THE STABILIZATION OF THE CATALYTIC TRANSITION-STATE OF THE SERINE-PROTEASE SUBTILISIN BPN'

The possible role of long-range electrostatic interactions on the catalytic activity of the serine protease subtilisin BPN' is investigated using protein engineering techniques. Charged residues on the surface of the enzyme some 13-15 angstrom from the active site were mutated to either neutral or oppositely charged residues. The effect of these mutations on the stability of a complex formed between subtilisin BPN' and Z-Ala-Ala-Pro-Phe-trifluoromethyl ketone, a transition-state inhibitor of the enzyme, was measured. The values of K(i) for the complex between the trifluoromethyl ketone and wild-type and mutant subtilisins were used to study the possible contribution of long-range electrostatics in stabilizing the charge distribution in the complex and thus, by analogy, on the transition state of hydrolysis for subtilisin BPN'. Measurement of k(on), k(off), and K(i) for the inhibition of wild-type and mutant subtilisins showed that charged mutations distant from the active site can affect k(off) and K(i) but have little effect on k(on). The experimental results show that there is a small, 0.10-0.46 kcal mol-1, but significant contribution to the binding energy from distant surface charges, at low ionic strength. The experimental results were compared to theoretical results, calculated using the DelPhi program for different charge distributions in the complex. The experimental results were found to be most consistent with a complex in which an ion pair is formed between the protonated active site histidine and the ionized oxyanion. Both experimental and theoretical results suggest that long-range electrostatic interactions do play a role in stabilizing the transition-state complex formed between enzyme and inhibitor. Although these effects are small, this suggests that long-range electrostatic interactions also play a role in stabilizing catalytic transition states. This maybe particularly important in enzymes which have a very asymmetric distribution of surface charge.