Gene M. Brown

[76] Dihydroneopterin aldolase from Escherichia coli

Publisher Summary This chapter discusses the preparation, purification, and properties of dihydroneopterin aldolase(H 2 -neopterin aldolase) enzyme. In assay method for preparation, the reaction is the removal, as glycolaldehyde, of two carbons of the three-carbon side chain of HE-neopterin. The product, glycolaldehyde, is not adsorbed to activated charcoal, whereas the other product, H 2 -pterin-CH 2 OH, and the residual unreacted substrate, H 2 -neopterin, are both adsorbed. Incubation of the enzyme, with H 2 -neopterin, labeled with tritium on carbon-3 of the side chain, followed by the measurement of the amount of radioactivity not adsorbed to activated charcoal (Darco G-60), is a measure of the amount of glycoaldehyde produced. The molecular weight of H 2 -neopterin aldolase is estimated at 100,000 by comparison of its migration during electrophoresis on SDS polyacrylamide gel with the migration of standard proteins. The pH optimum of the enzyme is at pH 9.6. The products of its action have been identified as H 2 -pterin-CH 2 OH and glycolaldehyde. The formation of products is linear, with incubation time up to 80 min, with the use of the purified enzyme (through the DEAE-cellulose step). The reaction is not reversible.

Nature of Multiple Forms of the Lactobacillus bulgaricus Factor (LBF).

Summary Treatment of pantethine with sodium sulfide, cysteine, glutathione, β-mercaptoethylamine, or several other thiols followed, in some cases, by oxidation with iodine resulted in formation of a series of new compounds with growth-promoting activity for Lactobacillus helveticus 80. These compounds appear to be mixed disulfides. The natural occurrence of similar mixed disulfides formed from pantetheine and other naturally occurring thiols may explain in part the presence of several chromatographically distinct forms of LBF in natural materials.

[193] Hydroxymethyldihydropteridine pyrophosphokinase and dihydropteroate synthetase from Escherichia coli

Publisher Summary Two enzymes present in Escherichia coli are discussed in the chapter catalyze the synthesis of dihydropteroic acid from hydroxymethyldihydropteridine, ATP (adenosine triphosphate) and p-AB. Two kinds of assays used are method A is used to measure only the activity of hydroxymethyldihydropteridine pyrophosphokinase and method B is used to measure the activity of either the pyrophosphokinase or the dihydropteroate synthetase. The cell-free systems from several different species of microorganisms use the chemically synthesized pyrophosphate compound for the synthesis of dihydropteroic acid in the absence of ATP. An intermediate compound formed in the presence of a purified enzyme from E. coli and show that the intermediate is identical with the chemically synthesized pyrophosphate ester. The pyrophosphate group is released by the substitution of p -AB during the second reaction. Crude cell-free extracts of E. coli B are prepared for GTP (guanosine triphosphate) cyclohydrolase. Polyacrylamide gel electrophoresis of the DEAE (diethylaminoethyl) eluate reveals the presence of only two significant protein bands, only one of which shows to possess hydroxymethyldihydropteridine pyrophosphokinase activity.

[90] Enzymic synthesis of 10-formyl-H4pteroyl-γ-glutamylglutamic acid from glutamic acid and 10-formyl-H4pteroylglutamic acid

Publisher Summary This chapter discusses the enzymic synthesis of 10-formyl-H 4 pteroyl-γ-glutamylglutamic acid from glutamic acid and 10-formyl-H 4 pteroylglutamic acid. Almost all the naturally occurring forms of folate are pteroylpolyglutamates, with the number of glutamate residues ranging from 2 up to 121 depending on the source. In Escherichia coli, the addition of the first glutamate residue is catalyzed by an enzyme named “dihydropteroate synthetase,” and the second glutamate residue is added in the presence of an enzyme. The activity of the enzyme can be assessed by incubation of the enzyme with [ 14 C]glutamic acid and 10-formyl-tetrahydrofolic acid (also called 10-formyl-tetrahydropteroylglutamic acid or 10-formyl-H 4 PteGlu), followed by the measurement of the amount of radioactivity that is rendered adsorbable to activated charcoal. Because the product of the action of the enzyme, 10-formyl-H 4 pteroyl-T-glutamyiglutamic acid [or 10-formyl-H 4 Pte(Glu) 2 ], is adsorbed to the charcoal, the amount of radioactivity adsorbed becomes a measure of the activity of the enzyme. This can be determined, by the elution of the product, from the charcoal, with ethanolic ammonia and the measurement of the amount of radioactivity in the eluate.