May 5, 10.58
COMMUNICATIONS TO THE EDITOR
We wish to report here the synthesis of the complex, uranyl bis- (sulfosalicylal)-0-phenylenediamine, which was prepared in pure crystalline form as the sodium salt, and is being applied as a heavy-atom dye in the crystal-structure analysis of the protein bovine pancreatic ribonuclease. The complex is taken up readily by crystals of ribonuclease from a 2.5 X M solution in 75 volume yo 2methyl-2,4-pentanediol. This complex is presumed to have the structure /="\
HC=N
N=CH
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of pyruvate' per minute per mg. protein a t 25' and pH 7.3. After incubation of the enzyme with I, the 2,4dinitrophenylhydrazone of pyruvic acid was isolated (m.p. 215"). The reaction is experimentally irreversible as demonstrated by the lack of incorporation of C1*into acid-stable linkages when Cl4O2 is incubated with the enzyme and either I or pyruvate. I n a preliminary report Eimhjellen2 has noted that oxalacetate (111) is formed by resting cells, and pyruvate by extracts, of an enterobacterium exposed to ADA. This finding would suggest the pathway shown C
I + CHI
The method of preparation involves simply the COOH mixing in aqueous solution of sulfosalicylaldehyde I I1 I11 (prepared according to Blau3), o-phenylenediamine, uranyl acetate and sodium hydroxide in the However, results with the purified enzyme from molar ratio 2 :1:1:2, and crystallization from Pseudomonas clearly eliminate free oxalacetate as an aqueous isopropyl alcohol. The analytical results TI I I I I for uranium are in accord with the proposed com: U, 30.3. position (Calcd. for C20H~2NzSzOl~NazU Found: U, 31.5).4 I n the absence of further ADA+ Prep+MDH ...... analytical data or degradative studies, the proposed structure cannot be proved, but is rendered highly probable by analogy with known complexes and $0.7 consideration of steric requirements. The assigned octahedral coordination of uranium (VI) is not the 0 most usual for this element, but has been observed d m previously in crystalline compounds, e.g., BaU04.6 lAcknowledgment.-The authors wish to a express appreciation to the several institutions the * continued support of which makes possible the work k 0 . 5 of the Protein Structure Project. The Dean Lang- v) z w muir Foundation, The Rockefeller Foundation, n Prep + The Damon Runyon Fund, The New York Foundation, Inc., The Polytechnic Institute of Brooklyn -a1 and The International Business Machines Cor- 0 Iporation.
+
1
L
(3) F. Blau, Monalsk., 18, 123 (1897). (4) Virginia Hong, Master's Thesis, Polytechnic Institute of Brooklyn, June, 19.57. ( 5 ) S. Samson and L. G. Sillen, A r k . f . Kem. M i x . och Geol.. 2 5 8 , KO. 21, 1 (1947).
CONTRIBUTION No. 15 FROM THE PROTEIN STRUCTURE PROJECT MURRAY VERNON KING POLYTECHNIC INSTITUTE OF BROOKLYN VIRGINIAHONGWEI 55 JOHNSON ST. BROOKLYN, NEW YORK RECEIVEDMARCH6 , 1958 ENZYMATIC UTILIZATION OF AN ACETYLENIC COMPOUND
Sir: A soluble enzyme system catalyzing the conversion of acetylenedicarboxylic acid (I) to equimolar quantities of pyruvic acid and carbon dioxide has been partially purified from sonic extracts of a species of Pseudomonas isolated from soil. The enzyme system has been purified 50-fold by protamine treatment, ammonium sulfate and acetone precipitation and by adsorption and elution from calcium phosphate gel. The most active preparations catalyze the formation of 55 pnioles
0.3
0
0.1
:\ I
I
0
I
1
I 2
M I N U T E S,
Fig. 1.-Tracing obtained from a Cary recording spectrophotometer of the rate of reduced pyridinenucleotide (DPNH) oxidation. All incubation mixtures contain 50 pmoles of phosphate a t pH 7.3 and 0.1 pinoles of DPNH per ml. When applicable the following were added to a total volume of 1.0 ml.: ADA, acetylene dicarboxylate, 10 pmoles, Prep, Pseudomonas enzyme, 2 pg.; 0'4, oxalacetate freshly prepared, 2 pmoles; LDH and MDH, an excess of lactic dehydrogenase and malic dehydrogenase, respectively. Arrows A and B denote the addition of substrate and oxalacetate, respectively. (1) Assayed by the method of T. E. Friedemann and G. E. Hsugen, J Bud. Chcm., 14'7, 415 (1943). (2) K. E. I3irnhjelleo. R i u c k e w . J , 6 4 , 4 p. ( 1 9 5 0 .
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COBIXUNICATIONS TO THE EDITOR
Vol.
so
TABLE; I intermediate on the following grounds : (1) oxalacetate as assayed by the method of Neish3 does not EFFECTOF BIOTIN-DEFICIENCY ON ENZYME ACTIVITY accumulate. ( 2 ) The oxalacetic decarboxylating Activity of cell-free extracts," pmoies of activity (both spontaneous and enzymatic) of product formed/mg. protein/hr. these preparations is too low to account for the Biotinrapid rate of pyruvate formation from I (Fig. 1). Enzyme system Control deficient ( 3 ) Addition of malic dehydrogenase to the incubaOrnithine carbamylatiou* 144 16 tion mixture (Fig. 1) indicates .the absence of free Aspartate ~ a r b a m y l a t i o n ~ 18 1.3 oxalacetate. These observations do not exclude cr-Ketoglutarate-aspartated 2.2 2.6 an enzyme-bound form of oxalacetate from partransaminase ticipating in the reaction as shown. a From L . a~abinosusgrown in ainirio acid medium1 cow The decarboxylation of I to form propiolic acid taining no arginine or uracil and 20 or 0.3 mpg./inl. of biotin, (CHiCCOOH) has been ruled out as the latter respectively. b Extract incubated with carbamyl phosphate, pmoles; tris-(hydroxymethy1)-aminomethane buffer, compound is not active in this system. The pos- 30 10 pmoles; ornithine, 10 pmoles in 1 ml. at pH 8.3 for 30 sibility of an enzyme-propiolate complex seems re- minutes at 35". Cirtulline determined colorimetrically.G mote as the formation of such a bound form might c ils in b with aspartate in lieu of ornithine; pH 7.5; incube expected to be reversible and result in the incor- bated at 25'; passed through a Dowex-50 column before * Determined as previdetermined' poration of radioactivity when C1402 was present. carbamylaspartate ously described.8 Further purification and studies on the mechanism ammonium sulfate fractionation (60 to 75y0satuof the reaction are in progress. ration) a t pH 7, heat treatment (65' for 5 minutes), ([i) W. J. P. h'eish, In D. Glick, "Methods of Biochemical Analysis," ammonium sulfate fractionation (50 to 7570 satuVal. 5. Interscience Publishers, Inc., New York, N. Y . , 1967. p. 168. (4) Fellow of T h e Jane Coffin Childs Memorial F u n d for Medical ration) a t p H 8.5; and chromatography on diethylResearch. This investigation has been aided by a g r a n t from T h e aminoethylcellulose gave a preparation with an Jaue roffin Childs Memorial F u n d for Medical Research. activity of 73,000 (pmoles citrulline produced,/mg. SATIONAL INSTITUTE OF ARTHRITIS protein,'hr.) . Assayed with Saccharomyces cereASD METABOLIC DISEASES IT.'. YAMADA4 visiae after hydrolysis with 3.6 N sulfuric acid, the NATIONAL INSTITUTES O F HEALTH ESTHER purified preparation and the original cell extract WILLIAM13. JAKOBY U. S. DEPARTMEST OF HEALTH EDUCATION A N D WELFARE (activity, 960) gave 0.1 and 3 mp g., respectively, BETHESDA14, MARYLAND of biotin/mg. protein, Unless biotin is present in RECEIVED MARCH 10, 1958 the enzyme in a form which does not yield an active form of biotin upon acid hydrolysis, biotin apparently is not a component of the enzyme but ROLE OF BIOTIN IN CARBAMYLATION REACTIONS exerts its effect during enzyme synthesis preSir: sumably in the formation of groups necessary for Biotin deficiency in Streptococcus lactis 8039 is the transfer of a carbamyl group. associated with a loss of ability to convert ornithine (6) R . M. Archibald, J . Bioi. Chem., 156, 121 (1944). and carbamyl phosphate to citrulline. Carbamyl ( 7 ) S. B . Koritz and P. P. Cohen, i b i d . , 209, 145 (1951) phosphate is involved in the conversion of aspartate ( 8 ) N. E. Tonhazy, K. G. White and n:. W. Umbreit, A r c h . I310 to N-carbamylaspartate, a precursor of pyrimi- chem. 28, 36 (1950). dines, and a study of biotin sulfone-inhibition of the CLAYTON FOUNDATION BIOCHEMICAL JOANNE X. RAVEL MARYLOUGRONA growth of Lnctobacillus arabinosus 17-5 suggested IXSTITCTE A N D THE DEPARTMEXT OF THEUTIVERSITYOF a possible role of biotin in pyrimidine biosynthesi~.~CHEMISTRY, J E A N WILLIAM S.HUXPHREYS SIIIVE In this investigation, a comparison of cell-free TEXAS,AUSTIX RECEIVED FEBRUARY 20, 1958 extracts of normal and biotin-deficient L. arnbinoSZLS revealed that the latter have a greatly diminished ability to carbamylate aspartate as well as DETERMINATION OF PROTON AFFINITY AND BOND ornithine4 as indicated in Table I. Specificity of DISSOCIATION ENERGY BY ION IMPACT METHOD this effect is indicated by the lack of an effect of Sir: biotin deficiency upon a-ketoglutarate-aspartate Until recently there were no experimental transaminase activity. Biotin or heat-inactivated methods for the determination of the proton affinity extracts of normal cells do not restore activity to of saturated molecules. A method for such a debiotin-deficient cell extracts. Normal activity termination has been proposed by the authors' cat1 be restored to biotin-deficient cells in a biotin and i t consists in investigating reactions between suppleniented growth medium in a few hours. ions and molecules in the ionization chainber of a Protein synthesis in the presence of biotin is es- mass spectrometer. If, under the esperiiiiental sential for the formation of the ornithine-citrulline conditions, a reaction can be observed, O I I C may enzyme iri biotiti. deficient cells of S. l a c t i ~ . Puri~ conclude that the reaction has no activation energy !jcation of this enzyme from extracts of S. lnctis by (to within an accuracy of 1-0 kcal./rnolt) :tnd that ~ 1 J) . A I . JSste.;, J. RI. l5
1.I
I
