However, the side chains on the leaving group of Ang I do not seem to be important for the selectivity of the HC to convert Ang I. Instead, the Lys40 side chain of the HC probably interacts with the negatively charged C-terminal carboxyl group of Ang I to Nilotinib stabilize the substrate. The Lys40 and Arg143 residues of the HC have previously been analyzed for their role in the selective Ang I conversion. In this study, Lys40 but not Arg143 was found to contribute to the high specificity of the HC to convert Ang I to Ang II. The mutants of the HC used in this analysis were Lys40Ala and Arg143Gln. The Arg143Gln mutant was actually shown to be more active than the wt in converting Ang I, indicating a minor role or no role at all of Arg143. The fact that mMCP-1, holding Lys143 and Met192 has a P2�� specificity for Leu, is a good Ang I converter further indicated a lack of function for Arg143 and Lys192 in Ang I conversion. The rat vascular chymase, with Arg143 and Thr192 residues thus not a predicted P2�� specificity for acidic aa residues, also has very good Ang I conversion capability. Interestingly, and in contrast to these predictions, we find that Lys192 but not Arg143 is of major importance for the cleavage rate of Ang I. Our results have previously clearly shown that both Arg143 and Lys192 residues of the HC are of major importance in mediating the specificity for acidic P2�� side chains of substrates. The marked difference in the importance of Arg143 and Lys192 in determining substrate specificity between peptides and long substrates is striking. This clearly shows the importance of analyzing a broad range of different substrates when DAPT looking for the natural in vivo substrates. This finding also applies to the screening for potent chymase inhibitors, as these low molecular weight compounds may have different binding characteristics from larger natural protein substrates for the HC. In conclusion, a more detailed knowledge of the specificity determining interactions may prove to be very valuable tools during the development of highly specific inhibitors of the HC and other medically important enzymes. The dependence of both Arg143 and Lys192 for the interaction with all five inhibitors tested in this study clearly points in this direction. A potentially very efficient way to obtain highly specific inhibitors could be to screen panels of inhibitors against both the wild-type and the mutant enzymes. This could highlight the maximum dependence of important residues in the specificity of the particular enzyme. Such inhibitors would have a good chance to primarily act on the enzyme of interest and leave related enzymes unaffected. Compared to work done in mouse pluripotent stem cells and human cancer cell lines, genome engineering in human pluripotent stem cells has been challenging partially due to low transfection/transduction efficiency and high apoptosis under stresses such as low-density plating, drug-selection and sorting.