Content area
Abstract
A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21.9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K^sub m^-200μM and 125μM, respectively) were bound to the enzyme through the linker (Asp)^sub 4^ Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K^sub m^=2.4μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding Avariation in the enzyme's uniquespecificity wasshown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466-800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These results suggest that the N-terminal fragment 118-465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.[PUBLICATION ABSTRACT]