both the specific nucleophilicity/electrophilicity character and the large relative difference of,3 pH units may likely play a role for the substrate specificity of hSCL. For a nucleophilic attack of C388 to occur using Cys as an electrophilic substrate, the active site C388 residue needs to be deprotonated to form a Cys-persulfide. For Sec as substrate, on the other hand, it would be expected that the protonation state of C388 will be less Calceolarioside-B critical, or even preferred to be in the protonated state, as the substrate itself is likely to be deprotonated and highly nucleophilic. In addition, a protonated C388 is clearly a better electrophile than a deprotonated C388, whereby the reaction would be expected to benefit from a more reactive substrate, such as Sec. Moreover, the completely conserved H145 and the positively charged ketimine nitrogen of the cofactor-substrate complex may be particularly effective to activate the Sec substrate because of the high polarizability of Se. Figure 6 illustrates the possible scenarios, for different substrates and the hSCL protein variants, in the step of the mechanism where C388 reacts with the substrate. Figure 6 is drawn based on the chemical mechanism involving elimination from the ketimine intermediate, originally proposed by Zheng et al.. However, the same reasoning is equally valid also for the alternative shorter mechanism with elimination directly from the quinonoid intermetiate. Hence, we propose that Sec specificity over Cys occurs in hSCL because C388 is maintained in its protonated form, thus only reacting when Sec is bound to the PLP. In addition, as described in the accompanying paper, group-I SCL/SD proteins contain a dynamic active site segment that houses the active site Cys residue. The location of D146 in relation to the dynamic active site segment also appears ideally suited to impose a second level of control. In the Eupalinilide-C closed conformation the sulfur atom of C388 is located,4A ? from D146, more or less in van der Waals contact, while in the open form this distance. A deprotonated, negatively charged, C388 is thus likely to shift the equilibrium towards the open or disordered state of the dynamic segment because of electrostatic repulsion from D146. This physico-mechanical mechanism is thus an additional and complementary way by which D146 may reduce the probability of positioning a deprotonated C388 in the closed conformation, as needed to react with Cys positioned into its substrate-binding cleft. Using the mechanisms described above as the basis for specificity should not yield a reaction that is strictly specific over infinite time scales because C388 would be deprotonated and located in the closed conformation a fraction of the time, depending on its local pKa, the pH, and the dynamics of the residue and active site domain.