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. 2014 Mar 5;53(15):2541–2555. doi: 10.1021/bi401671t

Figure 4.

Figure 4

Double-mutant cycle designed to test the roles of Met112 and Ala114 in positioning the general base. (A) Mutation of Asp38 to the “mispositioned” Glu general base allows it to populate alternate conformations (vertical arrows). Mutation of Ala114 affects the positioning of the general base starting from an enzyme with a positioned general base (top horizontal arrow) but has a smaller effect for an enzyme with a mispositioned general base (bottom horizontal arrow). For the sake of clarity, only the double-mutant cycle for Ala114 is shown. Effects on kcat for mutation of (B) Met112 to Ala and (C) Ala114 to Gly in KSI with a positioned Asp general base and a mispositioned D38E mutant general base. Values are from Table 3.