## 4 Discussion
Thus, the \(K_{\text{m}}\)/\(V_{\text{max}}\), \(k_{\text{cat}}\), and \(k_{\text{cat}}\)/\(K_{\text{m}}\) values are essentially changed, increasing \(K_{\text{m}}\)/\(V_{\text{max}}\) slopes and reducing the value range of turnover rates \(k_{\text{cat}}\) (1.9-3.9 \(\times\) 10 to 0.7-3.1 \(\times\) 10 s\({}^{-1}\)) and the substrate specificity constants \(k_{\text{cat}}\)/\(K_{\text{m}}\) (2.5-10.7 \(\times\) 10\({}^{3}\) to 0.5-8.3 \(\times\) 10\({}^{3}\) M\({}^{-1}\)s\({}^{-1}\)), at neutral pH 7.0. It is worth highlighting the low affinity shown for DL-glycerol-3-phosphate (0.5 \(\times\) 10\({}^{3}\) M\({}^{-1}\)s\({}^{-1}\)), which might suggest an essential role of the residues in the cap domain Phe-53 and Asn-54 or AtGpp counterparts Phe-43 and Asn-44 in the DL-glycerol-3-phosphate chemistry. Also, the degree of selectivity, defined by the ratio of \(k_{\text{cat}}\)/\(K_{\text{m}}\) between two different substrates [42], varied significantly. The AtSgpp3\(\Delta\) mutant displayed the highest level of discrimination with the D-ribose-5-phosphate versus DL-glycerol-3-phosphate, D-fructose-6-phosphate, D-glucose-6-phosphate, D-mannose-6-phosphate, and 2-deoxy-D-glucose-6-phosphate (\(k_{\text{cat}}\)/\(K_{\text{m}}\) ratio equal to 16.6, 6.9, 5.5, 3.0, and 1.7, respectively), compared to the normal protein (\(k_{\text{cat}}\)/\(K_{\text{m}}\) ratio equal to 3.5, 4.3, 3.1, 1.6, and 1.5, respectively). Promiscuous substrates are inadequately positioned relative to the catalytic machinery and therefore exhibit low \(k_{cat}\) values [38] and the \(k_{\text{cat}}\)/\(K_{\text{m}}\) ratio reflects both substrate binding affinity and proper orientation [42]. Taken together, these data suggest that reduction of cap flexibility in the AtSgpp3\(\Delta\) mutant may result in an improved substrate-binding affinity and proper orientation of D-ribose-5-phosphate in the active site compared to the other substrates used in this study. The pH dependence of the \(k_{\text{cat}}\)/\(K_{\text{m}}\) is derived from a composite of the ionization of several groups within the substrate and the enzyme [43]. As we previously discussed, in the case of AtSgpp, the pH-dependent reduced activity of this enzyme could be due to differences in side-chain protonation of the residues Lys-71 and His-72 in the cap domain [15; 19]. AtSgpp activity over D-ribose-5-phosphate was not significantly affected over the pH range assayed, which suggests a narrower chemoselectivity in the active site for this ligand compared to the remaining substrates tested. Not surprisingly, similar pH activity profile was observed for the mutant protein AtSgpp3\(\Delta\). This may be explained because the pH-activity profile is determined by the pKa values of the residues in the active site [44], which are not affected by the deletion of Phe-53, Asn-54, and Asn-55 in AtSgpp3\(\Delta\). Substrate ambiguity is a common feature amongst the members of the HAD superfamily, with 75% of the family members being able to metabolize more than five different substrates. Members with the lower degree of structural accessoration of the Rossmann fold (type C0, characterized for having minimal or no cap insertion) exhibit higher substrate specificity than those containing cap domain modules type C1, C2A, or C2B [45]. This observation is somehow intriguing based on the fact that cap domain contains the specificity determinants [20; 45]. However, enzymes are more amenable to evolution when the active site is composed of flexible loops, separated from a highly ordered core scaffold [14; 46] and accidental hydrogen bonds mediated by water molecules may play a role in promiscuous interactions [38]. Also, the flexibility of short loop regions might be a structural basis for recognizing distinct metabolites and has proved to be essential for discriminating structurally similar substrates [47].