FUNCTIONAL INTERACTION AMONG CATALYTIC RESIDUES IN SUBTILISIN BPN

Variant of the serine protease, subtilisin BPN′, in which the catalytic triad residues (Ser‐221, His‐64, and Asp‐32) are replaced singly or in combination by alanine retain activities with the substrate N‐succinyl‐L‐Ala‐L‐Ala‐L‐Pro‐L‐Phe‐p‐nitroanilide (sAAPF‐pna) that are at at least 103 to 104 above the non‐enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564–568, 1988]. A possible source of the residual activity was the hydrogen bond with the Nδ2 of Asn‐155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild‐type enzyme. Replacing Asn‐155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF‐pna by 150‐fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5‐fold greater than for the S221A enzyme. Thus, the catalytic role of Asn‐155 is dependent upon the presence of Ser‐221. The residual activity of the N155G:S221A enzyme (∼104‐fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. the mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor. These data suggest that Ser‐221 functions together with Asn‐155 to accelerate amide bond hydrolysis and that other transition state stabilizing interactions account for the residual rate enhancement of 103− to 104−fold. More generally, these studies illustrate the limitations of using site‐directed mutagenesis to probe the energetic importance of a single catalytic group whose function is dependent upon the interaction with others. Copyright © 1990 Wiley‐Liss, Inc.