Control of the rate limiting step by active site compactness: Reaction of UDPgalactopyranose mutase with UDP‐arabinopyranose

Abstract

Galactose is a common monosaccharide exclusively found in mammals as galactopyranose (Galp), the six‐membered ring form of this sugar. In Aspergillus fumigatus and mycobacteria, galactose in the cell wall is found as galactofuranose (Galf), the five membered ring form of this sugar. In these microorganisms, Galf biosynthesis begins with UDP‐Galp being converted to UDP‐Galf by UDP‐galactopyranose mutase (UGM). UGM is critical for the survival and proliferation of several prokaryotic and eukaryotic pathogens. Kinetic analyses have shown that bacterial UGM can also catalyze the isomerization of UDP‐arabinopyranose (UDP‐Arap). The possibility of using this sugar as a substrate for eukaryotic UGM has not been evaluated. Here, we probe the Galp‐Galf and Arap‐Araf isomerization catalyzed by Aspergillus fumigatus UGM using viscosity effects, solvent kinetic isotope effects, site directed mutagenesis, and computational biology. Kinetic studies show that eukaryotic UGM use UDP‐Arap/f as a substrate but with a much lower catalytic efficiency than UDP‐Galp/f. The data is consistent with a switch mechanism controlling the rate limiting steps, from the chemical step for UDP‐Galp to product release with UDP‐Arap or the Trp315Ala mutant. Computational studies suggest that tightness in the active site is responsible for this distinct behavior when either the substrate is changed or the side chain of Trp315 is deleted. These results provide important insights into understanding general enzyme catalysis and the UGM reaction mechanism, and will facilitate the development of inhibitors.Support or Funding InformationResearch reported in this publication was supported by grants from the National Institute of General Medical Sciences (NIGMS) award R01GM094469, the National Science Foundation award CHE‐1506206, and PICT 2436 préstamo BID. The authors are also very grateful to CONICET for financial support.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.