Vallejos-Baccelliere, G. | Search bioRxiv

bioRxiv2025

Unveiling the influence of salt concentration on the different stages of the catalytic cycle of a halophilic enzyme

Enzymes from halophilic organisms have adapted to function in salt concentrations near saturation, making them an interesting model for studying the effect of salt on enzyme catalysis. The main insights into the effect of ionic strength on enzyme catalysis come primarily from enzymes with positively charged surfaces interacting with negatively charged substrates (e.g., ribonucleases), whose activity decreases at high salt concentrations. In this study, we investigated the effect of salt on the kinetics of Glucose-6-phosphate dehydrogenase (G6PDH) from the halophilic archaeon Haloferax volcanii (HvG6PDH), which has optimal activity conditions at concentrations exceeding 2 M KCl. The enzyme catalyzes the NAD+-dependent oxidation of G6P, a negatively charged substrate, and glucose, a non-charged substrate. Using steady-state kinetics, we determined that the enzyme follows an ordered-sequential kinetic mechanism, with NAD+ being the first substrate to bind and NADH being the last released product. Through steady-state kinetic experiments, we found that the main effect of salt is on the KM for G6P, which decreased approximately 50-fold. For glucose dehydrogenase (glcDH) activity, the main effects were a 10-fold increase in kcat and a roughly 10-fold increase in kcat/KM for glucose. To analyze the effect of salt on the different stages of the catalytic cycle, we performed pre-steady-state experiments for both activities. We found that KCl did not affect the catalytic step in G6PDH activity, but it did increase the rate of catalysis in glcDH. Using a minimal model that accounts for substrate binding, chemical transformation, and product release, we determined that the main effect on G6PDH activity was an increase in the rate of G6P association. In contrast, for glcDH activity, the main effect was an increase in the rates of catalysis and product release. The results show that charge screening plays an essential role in the effect of salt on catalysis. Furthermore, it suggests differences in ion penetration to the active site between the two activities. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/632876v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@782511org.highwire.dtl.DTLVardef@1c9bce1org.highwire.dtl.DTLVardef@18f63faorg.highwire.dtl.DTLVardef@ecd1a9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

Glycogen phosphorylase from the methanogenic archaeon Methanococcus maripaludis: Unique regulatory properties of a pyridoxal 5'-phosphate independent phosphorylase

Glycogen phosphorylase (GP) is a critical enzyme in glycogen metabolism. Even though methanogens from the archaeal orders Methanosarcinales and Methanococcales are unable to grow on sugars, they store glycogen, which is metabolized through the glycogenolysis and glycolytic pathways when the carbon source for methanogenesis is depleted. Under these metabolic conditions, the activity of the GP enzyme is essential. To be active, all phosphorylases characterized to date require the cofactor pyridoxal 5-phosphate (PLP). This cofactor is covalently bound via Schiff base to a strictly conserved lysine residue at the active site. Extensive GP sequence analysis of organisms from different domains of life shows strict conservation of active site residues despite significant differences in sequence length. Interestingly, in GP sequences of organisms from the order Methanococcales of archaea, a threonine residue replaces the conserved lysine involved in PLP binding. The purification and characterization of recombinant GP from Methanococcus maripaludis show that the enzyme exhibits glycogen phosphorylase activity and high specificity for glycogen as a substrate. Analysis of the PLP content performed by several methods, such as absorbance, fluorescence, cyanohydrin adduct formation, and mass spectrometry, confirmed the absence of PLP. The results demonstrate that an archaeal GP from the order Methanococcales performs catalysis without the PLP cofactor, deviating from the well-established phosphorylase catalytic mechanism and revealing new scenarios for the glucosyltransferase reaction. Moreover, analysis of enzyme regulation shows that the activity is affected by various molecules, including nucleotides, intermediates of carbon metabolism, and phosphate species. Most of these molecules have not previously been identified as regulators of glycogen phosphorylases in prokaryotes. These results suggest that other GPs from Methanococcales can undergo complex regulation.

biochemistry↗