Vol. 76
3858
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COhIMUNICATIONS T O T H E EDITOR A RACEMASE FOR THREONINE I N ESCHERICHIA COLI
threonine and will not attack the L-allo, D- or Dallo forms. The conversion of L-threonine to DSir: threonine, followed by the disappearance of I,Growth studies with biochemically deficient threonine, appeared to occur a t the same rate as the mutants of yeast' and requiring threo- reverse transformation, nine have suggested that some strains possess a The presence of welchii deaminase during the threonine racemase. enzymatic racemization of D-threonine accelerated I wish to report the demonstration in a cell-free this conversion by removing L-threonine as it was extract from Escherichia coli of an enzyme capable formed. In such a system the disappearance of of transposing groups on both asymmetric carbon threonine and the appearance of a-ketobutyric acid atoms of threonine to convert D-threonine to L- could be followed by colorimetric estimation after threonine, for which ATP or yeast or muscle paper chromatography51~ (Table I ) . adenylic acid serves as a cofactor. I n rather Adenosine-3-phosphate stimulates racemization limited attempts it has not been possible to demon- as effectively as ATP and muscle adenylic acid. strate a vitamin B6requirement. The Christman synthetic coenzyme for aspartic Cells of Escherichia coli, strain K-12, grown on deaminase' has no effect. The quantity of enzyme the medium of Davis and Mingioli4 with 0.5% in K-12 is depressed sharply by the presence in glucose were dried in vacuo over phosphorus the growth medium of D-threonine. Fractionation pentoxide, ground with alumina and extracted with of the crude extract for more precise characteriza0.01 M phosphate buffer at pH 7.1. The activity tion of the enzyme is in progress. of the extracts toward threonine was determined ( 5 ) J. Awapara and B. Seale, J . Riol. Chem., 194, 447 (1952). after 16 hours dialysis against 0.01 2 2 1 phosphate ( 8 ) H. E. Umbarger and B. Magasanik, THISJ O U R N A L , 74, 4'253 buffer, pH 7.1, a t 2-3'. On storage the extracts (1952). (7) J. F. Christman, J . Bacfeuiol., 58, 565 (1949). lost the ability to deaminate L-threonine but retained that of converting L-threonine to the D- DEPARTMENT OF BACTERIOLOGY A N D IMMUNOLOGY form and vice versa. HAROLD AMOS HARVARD MEDICAL SCHOOL The formation of L-threonine starting from D- BOSTON,MASSACHCSETTS RECEIVED M A Y24, 1954 threonine was demonstrated by microbiological assay using a fastidious L-threonine-requiring mutant of Escherichia coli, llIL52, and quantitaTANTALUM SUBCHLORIDE' tively estimated by ammonia liberation with an L-threonine deaminase from Clostridium welclaii. S i r : The welchii enzyme proved to be specific for I,Among the subhalides mentioned in the literature, tantalum subchloride is one of the few whose TABLE I existence has neither been confirmed nor deCONVERSION OF D-THREONINE TO L-THREONIXE BY nied. According to Ruff and Thomas,2thermal deEXTRACTS OF Escherichia coli, STRAIX K-12 composition of the dichloride in vacuo leads to a T h e complete reaction system contained K-12 extract pyrophoric compound with the empirical formula (3.5 mg. extract protein), 50 &I of.D-threonine, 10 fig of Ta2C1. JVe have attempted to prepare this coinpyridoxal phosphate, welchii deaminase (2.4 mg. protein), pound by the procedure outlined by these authors. 5.0 pM. of A T P and 1.0 ml of 0.20 diphosphate buffer, pH Aluminum powder and tantalum pentachloride 7.8; incubed a t 37" for 3 hours in tubes flushed with nitrowere mixed in stoichiometric proportions according gen and stoppered; reaction stopped with 0.2 mi. of 5070 trichloroacetic acid and precipitate removed by centrifuga- to the equation tion; ammonia determined on supernatant b y microKjeldahl procedure. Reaction system
Complete Minus K-12 extract Minus D-threonine Minus A T P Minus pyridoxal phosphate Adenosine-5-phosphate for ATP Minus welchii deaminase
p l l . of h'Ha
10.2 0.2 1.3 3.8 10.5 10.7 6.4'
a At end of 3-hr. incubation a n aliquot was removed, from which K-12 enzyme was precipitated with 6 S HC1, precipitate removed by centrifugation, the supernatant returned t o pH 7.8 with WaOH and welchii enzyme added for 90 min. a t 37".
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(1) H. J. Tens, Oak Ridge National Laboratory, Report 164 (1948). (2) H. E. Umharger and E. A. Adelberg, J . Riol. Chem., 192, 883 (1951). (3) H. Amus aud C, S . Cohen, Biorhcm. J,,67, 338 (1854). (4) R. a.Dn*im tirid ii. S X r i n g b ! i , J . B a c t r r ~ c l .6',3, 17 (1960).
3TaClj
+ 2A1 = 2illC13 + 3TaC11
and heated in evacuated Pyrex tubes for 100 hours at 300 to 350". The aluminum trichloride was removed by sublimation, and the product, in all probability a mixture of TaCI4 and TaCl?, was transferred to small tantalum crucibles for subsequent thermal reduction. XI1 manipulations were made in dry argon. The X-ray powder diagram of the initial reduction product was extremely complex, but did not indicate the presence of aluminum, aluminum oxide or tantalum. Thermal decomposition of this product a t 600 to '700"produced a compound whose properties were identical to those reported by Ruff and Thomas for C
11) 1 iider research grant f r o m the National Krsedrch C orpordtiou, ,+mbridge hlass (2) 0 R u f f a n d P Thomrs, 8 anorp Chrm , 148, 1 (1925).
