Inhibition of glutamine synthetase by methionine sulfoximine. Studies on methionine sulfoximine phosphate.

The irreversible inhibition of glutamine synthetase by methionine sulfoximine is associated with the tight binding to the enzyme of adenosine diphosphate and methionine sulfoximine phosphate; the latter compound can be cleaved nonenzymatically and also by phosphatases to equimolar amounts of methionine sulfoximine and inorganic phosphate. Methionine sulfoximine phosphate is oxidized by L-amino acid oxidase to yield a phosphate-containing product. The chemical synthesis of methionine sulfoximine phosphate was achieved by treating methionine sulfoximine methyl ester with cyanoethyl phosphate and dicyclohexylcarbodiimide; the product was isolated from the reaction mixture and crystallized. The enzymeand chemically synthesized methionine sulfoximine phosphate products are identical as P revious studies in this laboratory (Ronzio et al., 1969a) have shown that the irreversible inhibition of glutamine synthetase from sheep brain by L-methionine sulfoximine in the presence of ATP and Mg2+ (or Mn2+) is associated with the tight binding to the enzyme of close to 8 moles each of meth* From the Department of Biochemistry, Cornell University Medical College, New York, New York 10021. Receitied February 25, 1969. Supported in part by the National Science Foundation and the National Institute of Health, U. S. Public Health Service. t Present address: Department of Biochemistry, University of Washington, Seattle, Wash. judged by paper chromatography in five solvent systems, paper electrophoresis at pH 2.8, 5.5, 7.5, and 9.0, relative stability a t various values of pH, and the rates of cleavage in 1 N HCI at 100' and by phosphatases. Treatment of methionine sulfoximine phosphate with hydrosulfite gave a product exhibiting the properties of methionine sulfimine phosphate, indicating that the phosphoryl moiety of methionine sulfoximine phosphate is attached to the sulfoximine nitrogen atom. L-Methionine sulfoximine phosphate inhibits glutamine synthetase in the absence of added nucleotide and metal ions; however, L-methionine sulfoximine phosphate inhibits the enzyme much more effectively in the presence of Mg2+ and adenosine diphosphate (and to a lesser extent adenosine triphosphate). ionine sulfoximine phosphate and ADP. It was established that the methionine sulfoximine phosphate derivative obtained from the enzyme (by brief heating at 100' or by treatment with perchloric acid) could be converted by treatment with acid or with several phosphatases into equimolar amounts of methionine sulfoximine and Pi. The present work, which was undertaken in order to further characterize the enzymebound methionine sulfoximine derivative, indicates that the phosphoryl moiety of this compound is attached to the sulfoximine nitrogen atom. The chemical synthesis of L-methionine sulfoximine phosphate has been achieved, and studies on the inhibition of glutamine synthetase by this compound have been carried out. 2674 R O W E , R O N Z I O , A N D M E I S T E R V O L . 8, N O . 6, J U N E 1 9 6 9 Experimental Section sulfoximine phosphate was found to be relatively stable to Materials. Glutamine synthetase was isolated from sheep brain as described by Ronzio et al. (1969b). L-Methionine sulfoximine, L-[' 4C, 2P]methionine sulfoximine phosphate (enzyme synthesized), phosphatases, ATP, ADP, and other compounds were obtained and characterized as stated previously (Ronzio et al., 1969a). Methionine sulfimine was prepared by treatment of methionine with hydrazoic acid (W. B. Rowe and A. Meister, 1969, in preparation). We are indebted to Dr. Daniel Wellner for the crystalline L-amino acid oxidase (Crotalus adamanteus), to Dr. Gerhard Schmidt for the yeast, prostate, and intestine phosphatases, and to Dr. R. T. Simpson and Dr. Bert L. Vallee for the Escherichia coli phosphatase. Dicyclohexylcarbodiimide was obtained from Schwarz BioResearch Inc. /3-Cyanoethyl phosphate was obtained as the barium salt from California Corp. for Biochemical Research. L-Ethionine sulfoximine was prepared as described by Bentley et al. (1951). Methods. Glutamine synthetase was determined by th.: y-glutamyl hydroxamate assay method (Wellner and Meister, 1966) using salt-free hydroxylamine. Paper electrophoresis was carried out as previously described (Ronzio et al., 1969a). In the experiments designed to determine ATP formation, 0.025 M sodium citrate buffer (pH 3.7) was used; at this value of pH AMP, ADP, ATP, and P, are separated. Paper chromatography (ascending) was carried out with the following solvents: solvent I, isobutyric acid-water-concentrated "?OH (66 : 33 : 1 , vlv); solvent 11, concentrated formic acid-sec-butyl alcohol-water (70:15:15, v/v); solvent 111, 9 5 x ethanol-1 N ammonium acetate (pH 5.5) (70:30, viv); solvent IV, n-butyl aicohol-acetic acid water (50:25 :25, v/v); and solvent V, r-butyl alcohol-methyl ethyl ketone-formic acid-water (40 : 30 : 15 : 15, viv). The RF values for methionine sulfoximine and methionine sulfoximine phosphate were, respectively, I , 0.64 and 0.26; 11, 0.13 and 0.09; 111, 0.37 and 0.14; IV 0.25 and 0.14; and V, 0.27 and 0.15. The position of P, and compounds containing phosphate was determined after chromatography by spraying the chromatograms with ammonium molybdate (Bandurski and Axelrod, 1952). Adenosine compounds were located by examining the chromatograms under ultraviolet light, and amino acids were located by spraying with ninhydrin. 14C and 32P were determined with a Nuclear-Chicago liquid scintillation counter using double-isotope technique. Liquid samples were counted in methyl Cellosolve-toluene ( 1 :2, viv). and 32P on dry Whatmann No. 3MM paper strips (0.5-1.0 cm wide X 1.8 cm long) were measured in a toluene solution containing 0.05 g of p-bis[2-(5-phenyloxazo1yl)lbenzene and 4 g of 2.5-diphenyloxazole per 1. Determinations were made with a precision of i 5 %. P, was determined by the Leloir and Cardini (1957) modification of the method of Fiske and Subbarow (1925) except as noted.