W. Z. Hassid

Formation and interconversion of sugar nucleotides by plant extracts

Abstract Extracts from mung bean seedlings and from a number of other plants contain a pyrophosphorylase (or pyrophosphorylases) capable of catalyzing the reversible formation of sugar nucleotides from UTP and a number of sugar 1-phosphates, according to the following reaction: UTP + S-1-P UDPS + PP. UDPG, UDPGal, UDPXy, and UDPAr are formed in this reaction from α- d -G-1-P, α- d -Gal-1-P, α- d -Xy-1-P, and α- and β- l -Ar-1-P, respectively. The reaction requires a bivalent metal ion (Mg ++ , Mn ++ , or CO ++ ). Sugar nucleotides are not formed from β- d -G-1-P, β- d -Gal-1-P, β- d -Xy-1-P, α- d -Ar-1-P, α-, β- l -Ar(F)-1-P, or α- d -Ar(F)-1-P. No exchange between PP and UTP 32 can be demonstrated except in the presence of one of the reactive sugar 1-phosphates. Mung bean seedling extracts also catalyze the interconversion of UDPG and UDPGal and of UDPXy and UDPAr.

Biosynthesis of Saccharides from Glycopyranosyl Esters of Nucleotides (“Sugar Nucleotides”)

Publisher Summary This chapter discusses the enzymic synthesis of numerous glycosides, including oligosaccharides and polysaccharides, which is subsequently effected by the transfer of a glycosyl residue from a saccharide donor to an appropriate acceptor. Transglycosylations involve only a redistribution of glycosidic linkages among saccharides, not a net increase in the number of such linkages. Another mechanism for the enzymic synthesis of saccharides is one that occurs through the transfer of the glycosyl group from a glycosyl phosphate to an appropriate acceptor. The evidence for the participation of glycosyl nucleotides in the biosynthesis of numerous glycosides, including disaccharides and polysaccharides, are described in the chapter.

Properties of a polygalacturonic acid-synthesizing enzyme system from Phaseolus aureus seedlings.

Abstract A cell-free enzyme system from Phaseolus aureus seedlings, which is capable of catalyzing the synthesis of polygalacturonic acid, was found to utilize UDP- d -galacturonic acid in preference to other nucleoside diphosphate d -galacturonic acid derivatives. It did not incorporate d -galacturonic acid residues from UDP-methyl- d -galacturonate into polygalacturonic acid. Maximum rate of polymerization occurs where preparations from seedlings germinated for 3 days are reacted with substrate at 30 °, pH 6, in the presence of 1.7 m m MnCl 2 and 0.4 m sucrose. The enzyme system has an apparent Michaelis constant of 1.7 × 10 −6 m , and at a substrate concentration of 3.7 × 10 −5 m will catalyze the polymerization of d -galacturonic acid residues at the rate of 4.7 mμmoles per milligram protein per minute. The enzyme is inactivated spontaneously, the rate of which is a function of temperature and pH. The addition of MnCl 2 delays inactivation.

Enzymic synthesis of uridine diphosphate glucuronic acid and uridine diphosphate galacturonic acid with extracts from Phaseolus aureus seedlings.

Abstract Extracts from mung bean seedlings contain a pyrophosphorylase (or pyrophosphorylases) capable of catalyzing the reversible formation of the following compounds: (a) uridine diphosphate d -glucuronic acid and inorganic pyrophosphate from uridine triphosphate and d -glucuronic acid 1-phosphate; and (b) uridine diphosphate d -galacturonic acid and inorganic pyrophosphate from uridine triphosphate and d -galacturonic acid 1-phosphate.

The enzymatic synthesis of a glucomannan

Abstract The mannose moiety of GDP- D -mannose- 14 C is transferred by a particulate enzyme from mung bean seedlings into a glucomannan. The product was characterized by isolation of a number of oligosaccharides after treatment of the product with a partially purified β-mannanase. Several of these oligosaccharides contained glucose and mannose in the approximate ratio of 1:1, 1:2 and 1:3 or 4, respectively. Since glucose- 14 C was found in the hydrolysis products of the oligosaccharides, it can be assumed that the particulate enzyme contains an epimerase that converts GDP- D -mannose- 14 C to GDP- D -glucose- 14 C.

Enzymic phosphorylation of d-glucuronic acid by extracts from seedlings of Phaseolus aureus

Abstract Enzyme preparations capable of phosphorylating d -glucuronic acid in the presence of ATP and MgCl 2 were obtained from mung bean seedlings. Both soluble and particulate preparations catalyzed the reaction. The enzymically formed phosphate ester was identified as α- d -glucuronic acid 1-phosphate.

The enzymatic synthesis of A (β-1-,2-)-linked glucan by an extract of Rhizobium japonicum

Abstract A particulate enzymatic preparation has been obtained from two strains of Rhizobium japonicum which synthetisized a labeled glucan from UDP-[ 14 C]glucose. The enzymatic system requires Mg 2+ or Mn 2+ and has an optimum activity at pH 7.5, and a K m for UDPG of 3·10 −4 M. This glucan can be found in the medium, after the growth of these bacteria in a synthetic medium containing d -glucose or d -galactose. It appears to be identical with a glucan previously isolated from cultures of Agrobacterium tumefaciens , by the criteria of the optical rotation, nuclear-magnetic-resonance spectroscopy and identification of the products of partial acidic hydrolysis. The main linkage between the glucosyl units is β-1,2. Some β-1,3 and β-1,6 linkages are also present. From the nuclear-magnetic-resonance spectrum, it can be deduced that the β-1,2 linkage accounts for more than 80% of the total bonds in the polyssaccharide. Influence of substitution at position 2 of glucose unit on the nuclear-magnetic-resonance spectrum is shown.

Metabolism of galactose in canna leaves and wheat seedlings

Abstract The transformation of randomly 14 C-labeled galactose was studied in Canna leaf disks during the course of a 3· hour respiration period in the dark. Introduction of this radioactive sugar into Canna leaf tissue caused a rapid appearance of 14 C-labeled sucrose and 14 C-labeled hexose monophosphates. When the radioactive sucrose was hydrolyzed to its monosaccharide constituents, the activity of the glucose was equal to that of fructose. The major proportion of the radioactive phosphorylated hexoses isolated from Canna leaf tissue, after infiltration of randomly 14 C-labeled galactose was an easily hydrolyzable galactose phosphate, which is probably galactose-1-phosphate. When galactose-1- 14 C was introduced into wheat seedlings and the glucose isolated from the sucrose after 2 hours was degraded to its individual carbon atoms, C-1 contained the major proportion of the activity (72%). C-6 contained practically all the remainder of the label (21%) which may have been produced through randomization with C-1 by a reversal of the glycolytic system in the respiring plant. The other four carbon atoms in the glucose chain possessed very little activity. The data indicate that galactose is converted directly to glucose without prior degradation of the chain, probably through a mechanism involving the enzyme, galactowaldenase.

Enzymatic synthesis of sucrose and other disaccharides.

Publisher Summary This chapter discusses enzymatic synthesis of sucrose and other disaccharides. The monosaccharides, D -glucose and D -fructose that are present in the plant as products of photosynthesis, are combined by an enzyme or enzymatic system, forming sucrose. As D -glucose and D -fructose may each exist in the α-and β-form, the following four different disaccharide configurations are possible when the two hexoses are combined: (1) α- D -glucose-β- D -fructose, (2) α- D -glucosea- D -fructose, (3) β- D -glucose-α- D -fructose, and (4) β- D -glucose-β- D -fructose. The enzymatic synthesis of sucrose also throws light on the formation of the furanose form of fructose in the sucrose molecule. The fact that sucrose is directly formed from D -glucose-l-phosphate and D -fructose supports evidence that the latter monosaccharide occurs in solution in an equilibrium mixture of furanose and pyranose forms. This makes it unnecessary to postulate a special mechanism of stabilization of a five membered (furanose) ring before the formation of compound sugars containing the D-fructose molecule. Another non-reducing disaccharide is synthesized because of enzymatic action of a P.saccharophila preparation on α- D -glucose-l-phosphate and L-araboketose.

Hydrolysis of amylose by β-amylase and Z-enzyme

Abstract 1. 1. The observation of Peat et al. (7) that potato amylose is incompletely hydrolyzed with pure β-amylase unless another factor (Z-enzyme) is added has been confirmed. 2. 2. Treatment with hot alkali of the resistant limit dextrin, obtained after hydrolysis of the amylose with β-amylase, has an effect similar to treatment with Z-enzyme in that it causes the residual dextrin to be further hydrolyzed with β-amylase. 3. 3. The exact degree of β-amylolysis depends on the plant material from which the sample of amylose is derived, on the particular subfraction used, or on the previous treatment of the amylose sample. Like natural amylose, synthetic amylose obtained by the action of potato phosphorylase on α- d -glucose 1-phosphate is not completely hydrolyzed by β-amylase. However, its limit of hydrolysis is considerably greater (89%, increased to 93% by the addition of emulsin). 4. 4. The Z-enzyme activity could be removed from the β-glucosidase and laminarase activities in almonds by purification, indicating that the obstacle to hydrolysis by β-amylase is probably not a β-glucosidic linkage. 5. 5. β-Glucosidase and laminarase are shown to be distinct from each other, as well as from Z-enzyme. 6. 6. The intrinsic viscosity of the limit dextrin is found to be approximately 11% lower than that of the parent amylose. This fact has been discussed with regard to the probable location of the anomalous linkage.