S. Kirkwood

UDPglucose dehydrogenase. Kinetics and their mechanistic implications.

Initial velocity and product inhibition studies were carried out on UDP-glucose dehydrogenase (UDPglucose: NAD+ 6-oxidoreductase, EC 1.1.1.22) from beef liver to determine if the kinetics of the reaction are compatible with the established mechanism. An intersecting initial velocity pattern was observed with NAD+ as the variable substrate and UDPG as the changing fixed substrate. UDPglucuronic acid gave competitive inhibition of UDPG and non-competitive inhibition of NAD+. Inhibition by NADH gave complex patterns.Lineweaver-Burk plots of 1/upsilon versus 1/NAD+ at varied levels of NADH gave highly non-linear curves. At levels of NAD+ below 0.05 mM, non-competitive inhibition patterns were observed giving parabolic curves. Extrapolation to saturation with NAD+ showed NADH gave linear uncompetitive inhibition of UDPG if NAD+ was saturating. However, at levels of NAD+ above 0.10 mM, NADH became a competitive inhibitor of NAD+ (parabolic curves) and when NAD+ was saturating NADH gave no inhibition of UDPG. NADH was non-competitive versus UDPG when NAD+ was not saturating. These results are compatible with a mechanism in which UDPG binds first, followed by NAD+, which is reduced and released. A second mol of NAD+ is then bound, reduced, and released. The irreversible step in the reaction must occur after the release of the second mol of NADH but before the release of UDPglucuronic acid. This is apparently caused by the hydrolysis of a thiol ester between UDPglucoronic acid and the essential thiol group of the enzyme. Examination of rate equations indicated that this hydrolysis is the rate-limiting step in the overall reaction. The discontinuity in the velocities observed at high NAD+ concentrations is apparently caused by the binding of NAD+ in the active site after the release of the second mol of NADH, eliminating the NADH inhibition when NAD+ becomes saturating.

The specificity of UDP-glucose 4-epimerase from the yeast Saccharomyces fragilis.

1. 1. The reactivity of UDP-glucose 4-epimerase (EC 5.1.3.2) from the yeast Saccharomyces fragilis toward 11 analogs of UDPG has been examined. The compounds tested involve stereochemical modifications of the hexosyl and ribosyl moieties of UDPG. 2. 2. Of the 11 compounds only UDP-β-l-arabinose, UDP-d-fucose and UDP-d-xylose are epimerized. However, evidence is presented indicating that these epimerizations are due to contaminating enzymes and are not due to UDPG 4-epimerase. 3. 3. The substances UDP-d-mannose, UDP-N-acetyl-d-galactosamine, UDP-d-allose and UDP-3-O-methyl-d-glucose were not epimerized. These observations are in agreement with the hypothesis of Budowskyet al.9 since the postulated hexose: uracil hydrogen bond, which is necessary for enzyme action in their mechanism, would be hindered in these substances. 4. 4. dUDPG is not acted upon by the yeast epimerase. It has been reported to serve as a substrate for the epimerase from calf liver. 5. 5. Other substrates which were not epimerized are UDP-β-d-glucose, UDP-d-glucuronic acid, UDP-4-O-methyl-d-glucose, ADPG, CDPG, GDPG, IDPG and dTDPG.

Studies on the structure and mechanism of an exo-(1ₒ3)-β-D-glucanase from basidiomycete qm806

Abstract A method for the large-scale production of a (1ₒ3)-β- D -glucan glucohydrolase (EC 3.2.1.58) from the culture filtrate of Basidiomycete QM806 is described. The final preparation is homogeneous by disc electrophoresis under non-dissociating and denaturing conditions, by ultracentrifugation, and by isoelectric focusing. Various physical and chemical characteristics of the enzyme have been determined, including terminal amino acid residues, extinction coefficient, and stability to pH extremes. The N-terminal amino acids are leucine and serine (Sangers method) and the C-terminal amino acids are alanine, serine, and glycine (hydrazinolysis). pH profile studies show that no group titrating in the region 2.5–8 is directly involved with substrate binding and that a single group having a p K a of 6.5 is involved in the catalysis. Photooxidation of the enzyme caused rapid inactivation. The pH-dependence of this photooxidation, and amino acid analysis of the photooxidized enzyme, indicate that decomposition of histidine is probably responsible for the loss of activity. Other chemical modifications performed were: treatment with hydrogen peroxide under acidic conditions, esterification with diphenyldiazomethane, and oxidation with N -bromosuccinimide. Oxidation with N -bromosuccinimide indicated that a tryptophan side-chain is involved in, but not necessary for, the catalytic activity.

The stereospecificity of hydrogen abstraction by uridine diphosphoglucose dehydrogenase

Abstract UDP-glucose dehydrogenase catalyzes the incorporation of tritium into UDP-glucose (UDPG) in the presence of UDP-α-D-gluco-hexodialdose (UDP-Glc-6-CHO) and [B- 3 H]-NADH. The 3 H is located exclusively at C-6 of the glucose moiety of UDPG and at least 79% of it is in the pro-R position. It is concluded that UDPG dehydrogenase catalyzes the abstraction of the pro-R hydrogen at C-6 of the glucose moiety of the substrate as the first step in the conversion of UDPG to UDP-glucuronic acid. The apparent lack of complete stereospecificity has been shown to result from a hitherto undetected reversible redox reaction prior to the release of UDP-glucuronic acid by the enzyme.

The stereospecificity of D-glucose-6-phosphate: 1L-myo-Inositol-1-phosphate cycloaldolase on the hydrogen atoms at C-6

D-Glucose-6-phosphate: 1L-myo-Inositol-1-phosphate cycloaldolase from rat testis or mammary gland removed stereospecifically the pro-S hydrogen atom at C-6 from D-glucose-6-phosphate. The pro-R hydrogen at C-6 remained in the product, 1L-myo-Inositol-1-phosphate and evidence is given that it is the hydrogen at C-1 of 1L-myo-Inositol-1-phosphate. The possible mechanism of cyclization is discussed.

Biosynthesis of the β-D-glucan of sclerotium rolfsii sacc. Direction of chain propagation and the insertion of the branch residues

Abstract The polysaccharide produced by the mold Sclerotium rolfsii Sacc. lends itself to studies of in vivo biosynthesis, since means are available to degrade pulse-labeled polysaccharide and determine the specific activity of all of the types of D -glucose residue present in its structure. Studies of this kind show, by direct measurement of the specific activities of the reducing and nonreducing terminals, that the direction of chain elongation is toward the nonreducing terminal. They show further that the interbranch, branch-point, and branch D -glucose residues are inserted into the structure of the molecule at about the same point in time, and that the D -glucose residues go intact into the structure, with no rearrangement of the carbon chain. The significance of these observations to the route of biosynthesis of the polysaccharide is discussed.

A vacuum system for the synthesis of nucleotide sugars on a micro scale

A glass vacuum apparatus and procedure are described for the synthesis, on a microscale, of nucleotide sugars. The apparatus is also a convenient way to prepare radioactive nucleotide sugars of high specific activity and in good yield. The apparatus has been employed in the synthesis of several novel compounds including UDP-d-fucose and UDP-4-O-methyl-d-glucose.

Carbohydrates from Precambrian and Cambrian Rocks and Fossils

Carbohydrate residues in eleven samples of early to late Precambrian rocks and fossils and one sample of Middle Cambrian Burgess Shale ranged from traces to more than 5 μg/g Free monosaccharides and a disaccharide, acid-extractable (polymeric) sugars, and recognizable polysaccharides were found in the samples. Geological conditions in the sampling areas suggest that the free sugars, being water-soluble, may not be indigenous to the rock but perhaps were introduced by ground-water circulation during the present or a preceding erosion cycle. Available evidence suggests that the acid-extractable monosaccharides and polysaccharides are at least partly native to the rocks or fossils in which they occur. The acid-extractable sugars obtained in these samples are β-D-galactose, β-D-glucose, mannose, arabinose, xylose, ribose, and rhamnose; the first two were identified enzymatically and they and the other sugars were also identified chromatographically. The polysaccharides found in these samples are linear α —1 →4 glucopyranose units suggesting starch, β — 1 → 4 glucopyranose units suggesting cellulose, and β — 1 → 3 glucopyranose unitssug-gesting laminaran. No starch residues were found n i the two lower Precambrian samples (Soudan and Coutchiching), but a trace of celluloseand laminaran was obtained in the Coutchiching. This may be an indication that cellulose-type structural polysac-charides and laminaran-type reserve sugars, but not starch-type food-reserve polysaccharides, existed in the early Precambrian.

Carbohydrate components of Paleozoic plants.

The residual monosaccharide components in aqueous and acid extracts of 25 species of Devonian-Permian plant fossils range from traces to 420 micrograms per gram. The species are distributed as follows: Pteridophyta-Psilophytales (2 species), Pteriodophyta-Equisetales (4 species), Pteridophyta-Lycopodiales (9 species), Pteridospermatophyta (6 species), Gymnospermae-Cordaitales (4 species).