Altering the binding determinant on the interdigitating loop of mandelate racemase shifts specificity towards that of D-tartrate dehydratase

The enolase superfamily (ENS) has served as a paradigm for understanding how enzymes that share a conserved structure, as well as a common partial reaction (i.e., metal-assisted, Bronsted base-catalyzed enol(ate) formation), evolved from a common progenitor to catalyze mechanistically diverse reactions. Enzymes of the mandelate racemase (MR)-subgroup of the ENS share interdigitating loops between adjacent, 2-fold symmetry-related protomers of the tightly associated homodimers that comprise their quaternary structures. For the MR-subgroup members MR and D-tartrate dehydratase (TarD), the tip of the loop contributes a binding determi-nant to the adjacent active site (i.e., Leu 93 and Lys 102, respectively). To assess the role of Leu 93 of MR in substrate specificity and catalysis, we constructed L93 variants bearing hydrophobic (L93A, L93F, and L93W), polar neutral (L93N), acidic (L93D), or basic (L93K and L93R) residues at position 93. Gel filtration-HPLC revealed that wild-type MR and all L93 MR variants, apart from L93R MR (dimeric), were tetrameric in solu-tion. The catalytic efficiency (k(cat)/K-m) was reduced in the R & RARR;S and S & RARR;R reaction directions for all variants, primarily due to reduced turnover (k(cat)). Substitution of Leu 93 by Lys or Arg to mimic Lys 102 of TarD enhanced the binding of malate and tartrate, with meso- and D-tartrate exhibiting linear mixed-type inhibition of L93K MR. Despite the striking 500-fold increase in the binding affinity of D-tartrate, relative to (S)-mandelate, L93K MR exhibited no TarD activity. MD simulations suggested that the failure of L93K MR to catalyze alpha-deprotonation (i.e., H-D exchange) arises from inappropriate positioning of the Bronsted base (Lys 166). Thus, a change in binding determinant on the interdigitating loop can play a significant role in governing substrate specificity within the ENS, but does not necessarily confer 'new' catalytic activity despite similarities in catalytic machinery.

Automated summary

The study demonstrates that the binding determinant on the interdigitating loop can play a significant role in governing substrate specificity and catalysis within the enolase superfamily. Various substitutions at position 93 of the MR enzyme resulted in changes in catalytic efficiency and substrate binding, but did not necessarily lead to the acquisition of new catalytic activities.