At the outer part of the active site in Trypanosoma cruzi glucokinase: The role of phenylalanine 337

The hole mutagenesis approach was used to interrogate the importance of F337 in Trypanosoma cruzi glucokinase (TcGlcK) in order to understand the complete set of binding interactions that are made by Dglucosamine analogue inhibitors containing aromatic tail groups that can extend to the outer part of the active site. An interesting inhibitor of this analogue class includes 2-N-carboxybenzyl-2-deoxy-Dglucosamine (CBZ-GlcN), which exhibits strong TcGlcK binding with a Ki of 710 nM. The residue F337 is found at the outer part of the active site that stems from the second protein subunit of the homodimeric assembly. In this study, F337 was changed to leucine and alanine so as to diminish phenylalanine's side chain size and attenuate intermolecular interactions in this region of the binding cavity. Results from enzyme - inhibitor assays revealed that the phenyl group of F337 made dominant hydrophobic interactions with the phenyl group of CBZ-GlcN as opposed to 7r - 7r stacking interactions. Moreover, enzymatic activity assays and X-ray crystallographic experiments indicated that each of these sitedirected mutants primarily retained their activity and had high structural similarity of their protein fold. A computed structure model of T. cruzi hexokinase (TcHxK), which was produced by the artificial intelligence system AlphaFold, was compared to an X-ray crystal structure of TcGlcK. Our structural analysis revealed that TcHxK lacked an F337 counterpart residue and probably exists in the monomeric form. We proposed that the D-glucosamine analogue inhibitors that are structurally similar to CBZ-GlcN may not bind as strongly in TcHxK as they do in TcGlcK because of absent van der Waals contact from residue side chains. (c) 2023 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Automated summary

The importance of F337 in TcGlcK was investigated using the hole mutagenesis approach, revealing its key interactions with aromatic tail groups in inhibitor molecules. Mutants retaining activity and structural similarity were obtained, suggesting the possibility of TcHxK existing as a monomer lacking the F337 residue. Similar D-glucosamine inhibitors may bind less strongly to TcHxK due to the absence of van der Waals contacts from residue side chains.