Isolation of 3-phosphohistidine from phosphorylated pyruvate, phosphate dikinase.

Abstract

(3,4) have demonstrated that the pyruvate,phosphate dikinase reaction occurs by a three-site nonclassical Tri (Uni Uni) Ping Pong mechanism. A pyrophosphoryl-and phosphoryl-histidyl residue of the enzyme has been proposed to serve as a carrier between the three sites (4).A number of enzymes that have reaction mechanisms involving phosphate moieties covalently bound to enzyme are now known (5). The phosphate has been found linked to proteins through different types of linkage, including a phosphoester with the hydroxyl group of serine (6), a phosphoramidate to N-1 or N-3 of histidine (7-10), and an acid anhydride to a y-carboxyl group of glutamic acid (11).The phosphoramidate and acid anhydride type of bond may be considered high in chemical energy (12), and thus either of these might meet the requirement of phosphoryl pyruvate, phosphate dikinase. However, at the outset we could not exclude the possibility of ester linkage to serine because the phosphate of this type of bond has been known to be transferable to ADP with concomitant formation of ATP (13). Thus, in an appropriate micro-environment even a seryl-phosphate bond may be considered a "high energy" type.In the present work using pyruvate,phosphate dikinase from Bacteroides symbiosus, the type of covalent bond of phosphoryl enzyme has been investigated and it has been shown to be a N-3-phosphohistidyl residue of the enzyme. (14). The product was purified by chromatography on a Dowex 1-X8 column (Cl-, 2 X 25 cm) (1). The radioactive fractions were pooled, lyophilized, and stored frozen. The concentration of P-enolpyruvate was determined enzymatically by a pyruvate kinase-lactate dehydrogenase system and specific radioactivity was calculated in cpm/,mol by dividing the cpm in material that migrated on paper chromatography with authentic P-enol[2-'4C]pyruvate by the Amols of P-enolpyruvate in the aliquot. The product was 92-94% pure. MATERIALS AND METHODS ChemicalsSynthesis of Phosphohistidine. Phosphohistidine was synthesized by reacting potassium phosphoramidate and histidine (15). Phosphoramidate was prepared from diphenylchlorophosphate (16). The phosphohistidine was purified by chromatography on a 2.4 X 30 cm column of Dowex 1-X8 using a 2-liter linear gradient of 0-1.0 M KHCO3, pH 8.25. The fractions were assayed for histidine (17) and phosphate (18), following cleavage of the phosphoramidate by acidification of an aliquot to pH 1.0. The fractions were pooled, cooled in ice, and brought to pH 3.0 with perchloric acid. After CO2 evolution ceased, the pH was rapidly adjusted to 9.0 with potassium hydroxide, and the sample was filtered to remove the precipitated potassium perchlorate and lyophilized. Upon rechromatography the product (with some potassium perchlorate) gave a single peak, which was 3-phosphohistidine, as confirmed by chromatography of the product with a tracer amount of a mixture of 1-and 3-phospho[carboxyl-'4C]histidine that had been prepared according to Hays et al. (10).Purification and Assay of Pyruvate,Phosphate Dikinase. The enzyme ...

Sequence and Structure Classification of Kinases

“…3 The mechanism of the phosphotransfer reaction in pyruvate phosphate dikinase involves the reversible phosphorylation of a histidine residue. 18,19 In this enzyme, the binding sites of the nucleotide and the pyruvate are in distant locations on the protein, and the shuttling of the phosphorylated histidine residue between the two active sites is accomplished by a dramatic swivel of the phospho-histidine domain. 20 Inspection of the multiple alignment of inositol 1,3,4-trisphosphate 5/6-kinase sequences has indicated that this kinase also belongs to the ATP-grasp family.…”

“…18,19 Although the mechanism utilized by protein kinases is currently a matter of debate, the phosphotransfer reaction in this family is thought to proceed either via a proposed simultaneous transfer mechanism, 15 which falls into the category of transition-state stabilization, or via acid -base catalysis. A second example includes the families of the ferredoxin-like fold group.…”

Electrochemical oxidation of enamines related to the key intermediate on thiamin diphosphate dependent enzymic pathways: evidence for one-electron oxidation via a thiazolium cation radical

“…The crude yellow product was chromatographed on 350 g of silica gel and eluted with ether-hexane (1:l): 'H NMR (400 MHz, CDC13-TMS) 6 The mixture was stirred 30 min and then a solution of CHJ (1.53 g, 10.77 mmol) in 5 mL of THF was added.…”

“…The resulting thick slurry was washed several times with anhydrous ether until compound lg, 0.6 g (81%), precipitated as a white powder: 'H NMR (200 MHz, Me2SO-TMS): 6 9.45. To a solution of compound 4g (0.5 g, 2.3 mmol) in 20 mL of anhydrous CH2C12 at 0 OC, was added (CH,)30BF4 (0.34 g, 2.3 mmol) in one portion.…”

“…The solution was Concentrated in vacuo to afford 11.2 g (91%) of 3g as an oil, which was crystallized from ether-hexane (9:l): 'H NMR (400 MHz, CDCI,-TMS) 6 4-Methylthiazole (6.0 mL, 60.0 mmol) in 5 mL of dry T H F was added dropwise to a solution of 60.0 mmol n-BuLi in 6.0 mL of THF at -78 "C under Ar.…”