Jan. 20, 1954
633
COMMUNICATIONS TO TIIE EDITOR
is also being studied. Some ostensible discrepancies have already been established as experimental errors, but all other reactions and other previously described formations of this compound seem to be compatible with the triazine formula I of C3H3N3.
is however highly significant and in accord with the reaction mechanism E
+ ATP 1_ E-P + ADP + Glu E-GlU -I-Pi + NH3 E + Glu-NHz
(1) (2) (31
E-P E-Glu
THEOHIO STATE UNIVERSITY RESEARCH FOUNDATION CHRISTOPH GRUNDMANN This formulation of reactions accounts for the large COLUMBUS 10, OHIO ALFREDKREUTZBERGER increase in exchange which occurs in the presence of RECEIVED DECEMBER, 2, 1953
ON THE MECHANISM OF THE ENZYMATIC SYNTHESIS OF GLUTAMINE'
Sir: It has now been established2v3that the synthesis of glutamine by plant enzymes proceeds according to the over-all reaction4 glutamic acid
+ NHI + ATP
glutamine
+ ADP +
Pi
The detailed mechanism by which this reaction proceeds, however, has hitherto remained obscure. Data bearing on this mechanism have been obtained by measuring the exchange of phosphate residues between Pi and ATP using the glutamine synthetase obtained from peas and purified 270-f0ld.~ The absolute amount of such exchange is low as shown in Table I. The way in which the exchange varies with the components of the reaction mixture
glutamate (line 3, Table I) since glutamate is an absolute prerequisite for exchange by this. mechanism. The fact that exchange is obtained a t all in the presence of only the enzyme, ATP and Pi is probably due to a small amount of ATPase known to be present in the enzyme preparation. The presence of ammonia or hydroxylamine (lines 4 and fi, Table I) results in the formation of glutamine or glutamylhydroxamic acid, thereby decreasing the amount of exchange of Pi b y keeping E-Glu a t a lower concentration. The results obtained would seem to preclude the possibility of a glutamyl phosphate acting as an intermediate in glutamine synthesis, since such a mechanism
+
Glutamate ATP Glutamyl phosphate NH, _c Glutamine Pi Glutamyl phosphate
+
+
would require that ammonia be present in order for exchange of Pi into A T P to take place. Similarly, the formation of an adenyl enzyme as recently imTABLE I plicated6 in the synthesis of acetyl-coA is not conPHOSPHATE EXCHANGE BETWEEN RADIOACTIVE INORGANIC sistent with the present results. ORTHOPHOSPHATE A N D A T P CATALYZED B Y GLUTAMINE The proposed mechanism for the synthesis of SYNTHETASE glutamine is also consistent with the suggestion of Reaction systems were incubated for 60 min. a t 23". All Elliott2 that glutamyl transferase activity and glusystems contained: 2.5 p moles P320r,2.0 p moles ATP, 20 tamine synthetase activity may both reside in a p moles MgS04, 10 p moles cysteine, and 45 p moles t n s (hydroxymethy1)-aminomethaneHC1 (pH 7.4). Additions single enzyme. Transferase activity would consist t o this basal mixture as noted above were 50 p moles gluta- simply of reaction no. 3 above. mate, 40 p moles XH3 (or NHzOH), and 0.5 mg. of enzyme Phosphorylation of the enzyme by ATP also has protein. The ATP and P, were separated chromatographibeen indicated as the initial step in the synthesis of cally by the procedure of Bandurski and Axelrod.8 glutathione from y-glutamylcysteine and glycine,' C.p.m./ml. Reaction system ATP Pi and in the synthesis of y-glutamylcysteine from 1 Noenzyme 120 900,000 glutamic acid and cysteine.8 The formation of a 2 Enzyme alone 1140 900,000 phosphorylated enzyme, therefore, may represent 3 Enzyme plus glutamate 9800 900,000 one general mechanism for the participation of A T P 4 Enzyme plus glutamate plus XHs 2600 900,000 in biological syntheses. 5 Enzyme plus glutamate plus NHzOH
740
900,000
(1) Supported in part by the Polychemicals Department, E. I. du Pont de Nemours and Co., and b y a grant-in-aid t o J.E.V. from the Charles F. Kettering Foundation. (2) W. H. Elliott, J. B i d . Chem., 201, 661 (1953). (3) George C. Webster, Plant Phrsiol., 28, 724 (1953). (4) Abbreviations used: ATP (adenosinetriphosphate), ADP (adenosinediphosphate), Pi (orthophosphate), E (enzyme), Glu (glutamate), Glu-NH2 (glutamine), CoA (coenzyme A). ( 5 ) R. S. Bandurski and B. Axelrod, J . Biol. Chcm., 193, 405 (1951).
KERCKHOFF LABORATORIES OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY GEORGE \ v E B S r E R PASADENA, CALIFORNIA J. E. VARNER 16, 1953 RECEIVED DECEMBER
e.
(6) Mary Ellen Jones, Fritz Lipmann, Helmut Hilz and Feodor 1.ynen, T x r s JOURNAL,76,3285 (1953). (7) John E. Snoke. i b i d . , 76, 4872 (1953). (8) George C. Webster and J. E. Varner, unpublished results.
