Stimulation Of Specific Activity Research Articles

Probing the dynamics between the substrate and the product towards glucose tolerance of Halothermothrix orenii β-glucosidase

Most β-Glucosidase (B8CYA8) are prone to inhibition by glucose. Experimentally observed specific activity of B8CYA8 on 20 mM, 50 mM, and 100 mM p-nitrophenyl-β-D-glucopyranoside (pNPGlc) substrate concentrations show surprise dependence on the presence of 0–3 M glucose at 335 K. We found that at high substrate concentration, the enzyme shows stimulation in specific activity with glucose and the glucose inhibition curve shifts toward the right with the increase in the substrate concentration. We employed atomistic molecular dynamics simulations of β-Glucosidase from Halothermothrix orenii at different glucose and pNPGlc concentrations to provide microscopic explanations to the experimentally observed non-monotonic glucose concentration dependence of the enzyme activity. Our results show that accumulation of substrate (pNPGlc) near the B8CYA8 catalytic site residues E166 and E354 and in the active site tunnel increases up to 0.5 M glucose when the specific activity is the highest. The number of pNPGlc in the tunnel decreases drastically when glucose concentration is more than 0.5 M, and hence the specific activity decreases. Potential of mean force (PMF) calculations showed that the most favorable interaction between pNPGlc and β-Glucosidase exists at 0.5 M glucose while at deficient and high glucose concentrations, the binding energy between the substrate and β-Glucosidase is very low. These studies provide the molecular basis towards understanding inhibition and stimulation of β-Glucosidase activity in the presence of glucose and may enable the optimum use of enzymes for the efficient conversion of high biomass loading saccharification reactions. Communicated by Ramaswamy H. Sarma

Elucidating the regulation of glucose tolerance in a β-glucosidase from Halothermothrix orenii by active site pocket engineering and computational analysis

β-Glucosidase catalyzes the hydrolysis of β-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of β-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with β-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a β-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity on p-nitrophenyl D-glucopyranoside stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the β-glucosidase at lower concentrations of glucose that, however, does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of β-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

Chemical chaperone rescue of mutant human cystathionine β-synthase

Missense mutations in the cystathionine beta-synthase (CBS) gene, such as I278T, are responsible for CBS deficiency, the most common inherited disorder in sulfur metabolism. Expression of human mutant CBS proteins in Saccharomyces cerevisiae reveals that most disease causing mutations severely inhibit enzyme activity and cannot support growth of yeast on cysteine-free media. Here, we show that the osmolyte chemical chaperones glycerol, trimethylamine-N-oxide, dimethylsulfoxide, proline or sorbitol, when added to yeast media, allows growth on cysteine-free media and causes increased enzyme activity from I278T and three other mutant CBS proteins. Rescuable mutants are ones that are predicted to cause a decrease in solvent accessible surface area. The increase in enzyme activity is associated with stabilization of the tetramer form of the enzyme. This effect is not specific to yeast, as addition of the chaperone glycerol resulted in increased I278T activity when the enzyme is produced either in Escherichia coli or in a coupled in vitro transcription/translation reaction. However, no stimulation of specific activity was observed when chaperones were added directly to purified I278T indicating that the presence of chemical chaperones is required during translation. We also found that by mixing different chaperones we could achieve rescue at significantly lower chaperone concentrations. Taken together, our data show that chemical chaperones present during the initial folding process can facilitate proper folding of several mutant CBS proteins and suggest it may be possible to treat some inborn errors of metabolism with agents that enhance proper protein folding.

The effect of high hydrostatic pressure on the modulation of regulatory enzymes from spinach chloroplasts.

High hydrostatic pressure enhanced the specific activity of regulatory enzymes of the Benson-Calvin cycle (fructose-1,6-bisphosphatase, glyceraldehyde-3-P dehydrogenase, phosphoribulokinase) which are modulated by the ferredoxin-thioredoxin system. High activity of chloroplast fructose-1,6-bisphosphatase required dithiothreitol, fructose 1,6-bisphosphate, and Ca2+. At 100 bar the A0.5 for fructose 1,6-bisphosphate (0.3 mM) was lower than that at 1 bar (1.5 mM), whereas similar variations of pressure did not alter the A0.5 for Ca2+ (55 microM). The response of chloroplast glyceraldehyde-3-P dehydrogenase exposed to 500 bar was a 4-fold increase in the NADP-linked activity; conversely, the NAD-dependent activity remained unchanged. The concerted action of high pressure and Pi (or ATP), both activators of chloroplast glyceraldehyde-3-P dehydrogenase, led to inactivation. On the other hand, the activity of phosphoribulokinase increased 10-fold when the enzyme was incubated at 1500 bar; the activation process was strictly dependent on the presence of dithiothreitol. At variance with these enzymes, bovine liver fructose-1,6-bisphosphatase, yeast glyceraldehyde-3-P dehydrogenase, and chloroplast ribulose 1,5-bisphosphate carboxylase, whose activities are not modulated by reduced thioredoxin, were inactivated by high pressure. The comparison of oligomeric enzymes revealed that the stimulation of specific activity by high pressure correlated with thioredoxin-mediated activation, and it did not depend on a particular subunit composition. Present results show that high pressure resembled thioredoxin, cosolvents, and chaotropic anions in its action on regulatory enzymes of the Benson-Calvin cycle. The comparison of physiological and non-physiological modulators suggested that thioredoxin-mediated modifications of noncovalent interactions is an important event in light-dependent regulation of chloroplast enzymes.