Hysteresis in human UDP-glucose dehydrogenase is due to a restrained hexameric structure that favors feedback inhibition.

Human UDP-α-d-glucose-6-dehydrogenase (hUGDH) displays hysteresis because of a slow isomerization from an inactive state (E*) to an active state (E). Here we show that the structure of E* constrains hUGDH in a conformation that favors feedback inhibition at physiological pH. The feedback inhibitor UDP-α-d-xylose (UDP-Xyl) competes with the substrate UDP-α-d-glucose for the active site. Upon binding, UDP-Xyl triggers an allosteric switch that changes the structure and affinity of the intersubunit interface to form a stable but inactive horseshoe-shaped hexamer. Using sedimentation velocity studies and a new crystal structure, we show that E* represents a stable conformational intermediate between the active and feedback-inhibited conformations. Because the allosteric switch occludes the cofactor and substrate binding sites in the inactive hexamer, the intermediate conformation observed in the crystal structure is consistent with the E* transient observed in relaxation studies. Steady-state analysis shows that the E* conformation enhances the affinity of hUGDH for the allosteric inhibitor UDP-Xyl by 8.6-fold (Ki = 810 nM). We present a model in which the constrained quaternary structure permits a small effector molecule to leverage a disproportionately large allosteric response.

Purification and kinetic properties of UDP-glucose dehydrogenase from sugarcane.

In this study, UDP-glucose dehydrogenase has been purified to electrophoretic homogeneity from sugarcane (Saccharum spp. hybrid) culm. The enzyme had a pH optimum of 8.4 and a subunit molecular mass of 52 kDa. Specific activity of the final preparation was 2.17 micromol/min/mg protein. Apparent K(m) values of 18.7+/-0.75 and 72.2+/-2.7 microM were determined for UDP-glucose and NAD(+), respectively. The reaction catalyzed by UDP-glucose dehydrogenase was irreversible with two equivalents of NADH produced for each UDP-glucose oxidized. Stiochiometry was not altered in the presence of carbonyl-trapping reagents. With respect to UDP-glucose, UDP-glucuronic acid, and UDP-xylose were competitive inhibitors of UDP-glucose dehydrogenase with K(i) values of 292 and 17.1 microM, respectively. The kinetic data are consistent with a bi-uni-uni-bi substituted enzyme mechanism for sugarcane UDP-glucose dehydrogenase. Oxidation of the alternative nucleotide sugars CTP-glucose and TDP-glucose was observed with rates of 8 and 2%, respectively, compared to UDP-glucose. The nucleotide sugar ADP-glucose was not oxidized by UDP-glucose dehydrogenase. This is of significance as it demonstrates carbon, destined for starch synthesis in tissues that synthesize cytosolic AGP-glucose, will not be partitioned toward cell wall biosynthesis.

Differential regulation of UDP-GlcUA transport in endoplasmic reticulum and in Golgi membranes.

BACKGROUND: In the endoplasmic reticulum (ER), the stimulation of UDP-glucuronosyltransferase (UGT) by UDP-GlcNAc is based on the interaction of transport across the ER membrane of UDP-GlcUA with UDP-GlcNAc. Intramicrosomal UDP-GlcNAc stimulates influx of UDP-GlcUA and thereby enhances delivery of UDP-GlcUA to the catalytic center of UGT in the ER lumen. AIM: The aim of this study is to investigate whether the interactions between nucleotide sugars for transport across the ER membrane also occur in the Golgi apparatus, and thereby affect UGT activity in Golgi membranes. RESULTS: We found that Golgi membrane preparations display UGT activity which, unlike in ER membranes, is not stimulated by UDP-GlcNAc. Efflux of intravesicular UDP-GlcNAc and UDP-Xyl marginally enhanced uptake of UDP-GlcUA in Golgi vesicles; such trans-stimulation was much more pronounced in the ER. Efflux of intravesicular UDP-GlcNAc was strongly trans-stimulated by cytosolic UDP-GlcUA in ER-derived vesicles but less so in Golgi-derived vesicles. CONCLUSION: The interaction between transport of UDP-GlcUA and transport of UDP-GlcNAc or UDP-Xyl is different in Golgi vesicles compared with ER vesicles. This finding is consistent with the different effects of UDP-GlcNAc on glucuronidation in Golgi and ER.

Uridine diphosphoxylose enhances hepatic microsomal UDP-glucuronosyltransferase activity by stimulating transport of UDP-glucuronic acid across the endoplasmic reticulum membrane.

The UDP-glucuronosyltransferase (UGT) system fulfils a pivotal role in the biotransformation of potentially toxic endogenous and exogenous compounds. Here we report that the activity of UGT in rat liver is stimulated by UDP-xylose. This stimulation was found in native microsomal vesicles as well as in the intact endoplasmic reticulum (ER) membrane, as studied in permeabilized hepatocytes, indicating the potential physiological importance of UDP-xylose in the regulation of UGT. We present evidence that UDP-xylose enhances UGT activity by stimulation of (i) the uptake of UDP-glucuronic acid across the ER membrane and (ii) the elimination of the UDP and/or UMP reaction product out of the ER lumen. UDP-xyloe produced a marked trans-stimulation of microsomal UDP-glucuronic acid uptake when it was present within the lumen of the ER. When UDP-xylose was presented at the cytosolic side of the ER, it acted as a weak inhibitor of UDP-glucuronic acid uptake. Likewise, cytosolic UDP-glucuronic acid strongly trans-stimulated efflux of intravesicular UDP-xylose, whereas cytosolic UDP-xylose was inefficient in trans-stimulating efflux of UDP-glucuronic acid. Microsomal UDP-xylose influx was markedly stimulated by UMP and UDP. Such stimulation was only apparent when microsomes had been preincubated and thereby preloaded with UMP or UDP, indicating that UMP and UDP exeted their effect on UDP-xylose uptake by trans-stimulation from the luminal side of the ER membrane.

Glucomannan synthesis in pea epicotyls: the mannose and glucose transferases.

Membrane fractions and digitonin-solubilized enzymes prepared from stem segments isolated from the third internode of etiolated pea seedlings (Pisum sativum L. cv. Alaska) catalyzed the synthesis of a beta-1,4-[14C]mannan from GDP-D-[U-14C]-mannose, a mixed beta-1,3- and beta-1,4-[14C]glucan from GDP-D-[U-14C]-glucose and a beta-1,4-[14C]-glucomannan from both GDP-D-[U-14C]mannose and GDP-D-[U-14C]glucose. The kinetics of the membrane-bound and soluble mannan and glucan synthases were determined. The effects of ions, chelators, inhibitors of lipid-linked saccharides, polyamines, polyols, nucleotides, nucleoside-diphosphate sugars, acetyl-CoA, group-specific chemical probes, phospholipases and detergents on the membrane-bound mannan and glucan synthases were investigated. The beta-glucan synthase had different properties from other preparations which bring about the synthesis of beta-1,3-glucans (callose) and mixed beta-1,3- and beta-1,4- glucans and which use UDP-D-glucose as substrate. It also differed from xyloglucan synthase because in the presence of several concentrations of UDP-D-xylose in addition to GDP-D-glucose no xyloglucan was formed. Using either the membrane-bound or the soluble mannan synthase, GDP-D-glucose acted competitively in the presence of GDP-D-mannose to inhibit the incorporation of mannose into the polymer. This was not due to an inhibition of the transferase activity but was a result of the incorporation of glucose residues from GDP-D-glucose into a glucomannan. The kinetics and the composition of the synthesized glucomannan depended on the ratio of the concentrations of GDP-D-glucose and GDP-D-mannose that were available. Our data indicated that a single enzyme has an active centre that can use both GDP-D-mannose and GDP-D-glucose to bring about the synthesis of the heteropolysaccharide.

A xylosyltransferase involved in the synthesis of a protein-associated xyloglucan in suspension-cultured dwarf-French-bean (Phaseolus vulgaris) cells and its interaction with a glucosyltransferase.

A particulate enzyme preparation made from suspension-cultured dwarf-French-bean (Phaseolus vulgaris) cv. Canadian Wonder cells was shown to incorporate xylose from UDP-D-[14C]xylose into polysaccharide. The reaction was dependent upon the presence of UDP-D-glucose and was stimulated, and apparently protected, by GDP-D-glucose and GDP-D-mannose, though neither was able to replace UDP-D-glucose as a glycosyl donor. The product of the reaction was identified as xyloglucan by analysis of products of enzyme breakdown and acid hydrolysis. Mr determination after proteinase K digestion indicated that the nascent xyloglucan is closely associated with protein. Preincubation of the enzyme with UDP-D-glucose stimulated incorporation from UDP-D-[14C]xylose, suggesting an 'imprecise' mechanism of biosynthesis, as defined by Waldron & Brett [(1985) in Biochemistry of Plant Cell Walls (Brett, C. T. & Hillman, J. R., eds.) (SEB Semin. Ser. 28), pp. 79-97, Cambridge University Press, Cambridge].

Regulatory mechanisms of UDP-glucuronic acid biosynthesis in cultured human skin fibroblasts.

Kinetic parameters and regulatory properties of UDPGDH extracted from cultured human skin fibroblasts were determined and compared with those of UDPGDH from cornea and epiphysial-plate cartilage. Fibroblast enzyme showed an affinity for UDPG 7 times higher than cartilage enzyme and 42 times higher than cornea enzyme. UDP-xylose acted as a co-operative allosteric inhibitor, but under the same experimental conditions fibroblast enzyme was significantly less inhibited. These results were in agreement with the different GAG production of the cells we studied. Fibroblast UDPGDH activity was regulated by the NAD/NADH ratio and it was also affected by modifications of extracellular matrix composition. A significant increase of UDPGDH affinity for UDPG was observed after the treatment of the monolayers with Chase ABC.

A glucuronyltransferase involved in glucuronoxylan synthesis in pea (Pisum sativum) epicotyls.

A particulate enzyme preparation made from epicotyls of 1-week-old etiolated pea (Pisum sativum) seedlings was shown to incorporate glucuronic acid from UDP-D-[U-14C]glucuronic acid into a hemicellulosic polysaccharide. Optimum conditions for the incorporation include the presence of Mn2+ ions at between 4 and 10 mmol/litre and a pH between 5 and 6. UDP-D-xylose at 1 mmol/litre allows incorporation to continue for at least 8 h. In its absence, the reaction stops within 30 min. Analysis of the product by partial and total acid hydrolysis, followed by paper chromatography or electrophoresis, indicates that the polysaccharide produced is a glucuronoxylan.

Enzymatic formation of UDP-N-acetylgalactosamine in epiphysial-plate cartilage.

The activity of UDP-N-acetylglucosamine 4'-epimerase (EC 5.1.3.7) from newborn pig epiphysial-plate cartilage was investigated. The formation of radioactive UDP-N-acetylgalactosamine from UDP-N-acetyl[U-14C]-glucosamine was demonstrated by radioautography, after hydrolysis of UDP-derivatives and separation of the hexosamines by paper chromatography. The pH optimum and the Km values for UDP-N-acetylglucosamine and NAD were determined. At equilibrium, the ratio UDP-N-acetylglucosamine/UDP-N-acetylgalactosamine reaches a value of about 2.3. The effect of UDP-xylose and UDP-glucuronic acid on the enzyme activity was investigated. NADH inhibits UDP-N-acetylglucosamine 4'-epimerase activity. The inhibitory effect of NADH seems to be strikingly correlated with the value of NAD/NADH ratio and pH.

Effect of some nucleotides on the regulation of glycosaminoglycan biosynthesis.

The effect of some nucleotides on UDP-glucose dehydrogenase (EC. 1.1.1.22) and UDP-glucose 4'-epimerase (EC 5.1.3.2) extracted from epiphysial-plate cartilage of newborn pigs was investigated. UDP-xylose acts as a co-operative allosteric inhibitor of UDP-glucose dehydrogenase, whereas it does not inhibit UDP-glucose 4'-epimerase activity: the inhibition of UDP-glucose dehydrogenase results in an increase of UDP-galactose synthesis, in agreement with the equilibrium constant of UDP-glucose 4'-epimerase reaction. Because of the presence of UDP-glucose 4'-epimerase activity in the enzyme extract, the addition of UDP-galactose induces an increase in reaction rate of UDP-glucose dehydrogenase. NADH inhibits both UDP-glucose dehydrogenase and UDP-glucose 4'-epimerase activities: in the presence of non-saturating NAD concentrations, NADH acts as a co-operative allosteric inhibitor of both enzymes. The inhibitory effect of NADH seems to be strikingly correlated with the value of NAD/NADH ratio and pH. In any case, the percentage inhibition of UDP-glucose 4'-epimerase, under the same experimental conditions, is always higher than that of UDP-glucose dehydrogenase.

Interactions of urdine diphosphate glucose dehydrogenase with the inhibitor urdine diphosphate xylose.

1. UDP-xylose and UDP-glucose both bind to UDP-glucose dehydrogenase in the absence of NAD+, causing an enhancement of protein fluorescence. 2. The binding of UDP-xylose is pH-dependent, tighter binding being observed at pH8.2 than at pH8.7. 3. At low protein concentrations sigmiodal profiles of fluorescence enhancement are obtained on titration of the enzyme with UDP-xylose. As the protein concentration is increased the titration profiles become progressively more hypebolic in shape. 4. The markedly different titration profiles obtained on titrating enzyme and the enzyme-NAD+ complex with UDP-xylose suggests a conformational difference between these two species 5. NAD+ lowere the apparent affinity of the enzyme for UDP-xylose. 6. There is no change in the apparent moleculare weight of UDP-glucose dehydrogenase on binging UDP-xylose. 7. Protein modification by either diethyl pyrocarbonate or 5, 5'-dithiobis-(2-nitrobenzoate) does not "desensitize" the enzyme with respect to the inhibition by UDP-xylose. 8. UDP-xylose lowers the affinity of the enzyme for NADG. 9. It is suggested that UDP-xylose is acting as a substrate analogue of UDP-glucose and causes protein-conformational changes on binding to the enzyme.