Exploring the active site of tripeptidyl-peptidase II through studies of pH dependence of reaction kinetics

► The first system for purifying mammalian TPP II expressed in E. coli is presented. ► Through determination of pH-dependence the active site of TPP II is investigated. ► The pH-dependence of kcat/KM is affected by substrate protonation state. ► Evidence for non-productive binding of some substrates in TPP II is revealed. ► The microenvironment in the active site is more charged in insect TPP II.