Adenosine 5'-O-([gamma-18O]gamma-thio)triphosphate chiral at the gamma-phosphorus: stereochemical consequences of reactions catalyzed by pyruvate kinase, glycerol kinase, and hexokinase.

Abstract

Our understanding of the molecular details of enzyme-catalyzed phosphoryl group transfer processes is still rudimentary, despite the importance of phosphokinases in the synthesis and utilization of ATP. The methods available for mechanistic investigation of these reactions have not provided unambiguous answers to such fundamental questions as whether covalent phosphoryl-enzymes are kinetically obligatory (1) and how the rates of phosphoryl group transfers are accelerated (2). We report here a stereochemical approach to these problems, and present evidence that three kinases [pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40), glycerol kinase (ATP:glycerol 3-phosphotransferase; EC 2.7.1.30), and hexokinase (ATP:hexose 6-phosphotransferase; EC 2.7.1.1)] not only catalyze the phosphoryl transfers to ADP with complete stereospecificity, but that each also follows the same stereochemical course (i.e., retention or inversion) with respect to the chirality of the phosphoryl group transferred. EXPERIMENTALThe 2-and 3-[180]phosphorothioate esters of D-glycerate, chiral at phosphorus, were prepared as shown in Figs. 1 and 2. All reactions were monitored by thin-layer chromatography, and the purified products were characterized by 31P nuclear magnetic resonance, by gas chromatography and mass spectrometry of the trimethylsilyl derivatives, and by enzymic assay where appropriate. All enzymes used were of the highest grade available from Sigma, and were dialyzed against 100 mM triethanolamine-HCl buffer, pH 7.5. D-Glyceric acid was converted into its methyl ester by using diazomethane, and a mixture of the ester 1 and ethanol was allowed to react with PC13 in anhydrous benzene. After workup, the cyclic hydrogen phosphite was treated with sulfur/triethylamine (3, 4) to give the pair of diastereoisomers 2. On treatment with aqueous triethylamine these compounds were converted into the diastereoisomers of the cyclic 2,3-phosphorothioate of D-glycerate (3a and 3b) in 48% yield from 1. (The methyl ester hydrolyzes under these mild conditions, presumably catalyzed by the neighboring phosphorothioate.) The diastereoisomers of 3a and 3b were separated by careful ion-exchange chromatography on DEAE-cellulose; each diastereoisomer was isolated in greater than 98% purity. This separation was conveniently monitored by gas chromatography of the trimethylsilyl derivatives.Hydrolytic ring opening of an isolated diastereoisomer of 3 (whether the diastereoisomer used was actually 3a or 3b is unknown, but for the purposes of illustration, Fig. 2 shows 3a) by Li18OH [approximately 1 M, prepared from metallic lithium and H2180 (97.3% atom excess 180)] at room temperature for 3 hr yielded a mixture of the 2-and 3-phosphorothioates of D-glycerate (4 and 5) in 75:25 ratio, as estimated by gas chromatography of the trimethylsilyl derivatives. This mixture was subjected to the following conversions (see Fig. 3).Route z. Treatment of the mixture (of 4 and 5) with sodium acetate buffer at pH 5.4 caused the preferential loss of sulfur ...

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

“…The synthesis of adenosine-5'-0( 7-thio) triphosphate with a chiral y-phosphate was synthesized enzymatically using thiophospho-enolpyruvate and ADP with pyruvate kinase. The resulting ",/-chiral ATP-7-~80-,,/-S is utilized as a substrate by hexokinase and by glycerol kinase and the stereochemical analysis of the products show that these three kinases catalyze phosphoryl transfer reactions with the same stereochemical path (87). The subsequent synthesis and utilization of the chiral 170, 180] ATP of a fixed configuration (S) was used to determine the absolute stereochemical path of phosphoryl transfer for hexokinase and glycerol kinase.…”

Interaction of sn-glycerol 3-phosphorothioate with Escherichia coli: effect on cell growth and metabolism

“…We have investigated the stability of glycerolphosphorothioate in aqueous solution as a function of pH. Although phosphorothioate monoesters are relatively stable compounds, under certain conditions they can undergo acid-catalyzed desulfurization reactions, giving rise to their parent "all-oxy" derivatives (23). In a typical experiment, [1,3-3H]glycerol-phosphorothioate (100,000 cpm) was incubated with 100-,u portions of various buffers at pH 3 to 7 for 16 h at 30°C.…”

The mitochondrial protein import motor: Dissociation of mitochondrial hsp70 from its membrane anchor requires ATP binding rather than ATP hydrolysis

“…This concern is particularly relevant in the case of ATPyS, which is a substrate for some ATPases (Eckstein, 1983;Turina & Capaldi, 1994). For example, ATPyS can be hydrolyzed by hexokinase, glycerol kinase, and actin (Orr et al, 1978;M.-F. Carlier, pers. comm.…”