FORMATION OF GUANOSINE DIPHOSPHATE FUCOSE FROM


Enzymatic Evidence for the Configuration of d-Isocitric Acid. ALTON MEISTER , MARYELLEN STRASSBURGER. Nature 1963 200 (4903), 259-260 ...
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Iiuried in the electron density of the cobalt atom as has been suggested' previously whereas these numerically accurate calculations provide no support for a Co-H distance of -2.0. DEPARTMEST O F CHEMISTRY MASSACHUSETTS INSTITUTE O F CANBRIDGE 39, MASS

TECHNOLOGY F.A.

RECEIVED JULY

COTTON

7, 1958

FORMATION O F GUANOSINE DIPHOSPHATE FUCOSE FROM GUANOSINE DIPHOSPHATE MANNOSE

Sir: A fucose containing nucleotide, guanosine diphosphate fucose, has been isolated recently from sheep milk' and from Aerobacter aerogenes.2 I t has now been found that GDPM3can be converted to guanosine diphosphate fucose by dialysed crude extracts of A . aerogenes4 in the presence of TPNH. The conversion was detected as follows: GDPAI, prepared from yeast,5 was incubated with T P N H and crude bacterial extracts obtained by shaking the cells with glass beads6 followed by centrifugation and dialysis. The guanosine sugar nucleotides were then isolated and purified from the incubation mixtures by charcoal adsorption and paper ~ h r o m a t o g r a p h y . ~Chromatography of the sugar liherated by 0.01 N HCl hydrolysis of the isolated nucleotides revealed, in addition to mannose, the presence of a second compound. This new compound exhibited the characteristic 400 mp absorption peak when examined by the specific colorimetric assay for 6-deoxyhexose.' Upon paper chromatography, the unknown sugar co-chromatographed with authentic fucose using the solvents 2TABLE I COYVERSIONOF GUANOSINE;DIPHOSPHATEMANNOSE TO GUANOSISF: DIPHOSPHATE FUCOSE The reaction mixtures contained 1.0 pmole G D P M , 3 mg. of crude extract protein and additions in 1.0 ml. of 0.05 M tris-(Ii?-droxj~meth~I)-aminomethane buffer, p H 7.8. Incuhation was carried out a t 37' for 4 hours. T h e nucleotides were then adsorbed on charcoal and the nucleotide 1)nunrl sugars liherated by heating for ten minutes a t 100" i n 0.01 .V H C 1 . Xfter deionization with Amberlite MR-3, tlic fucose in the hydrolysate was estimated colorimetrically7 n r b y paper chromatography. Additions

&mole fucose formed


Sl~llt.

2.0 pinoles T P N 0.0 prnoles TPXIT 2 0 prnoles DPSI-I 2 . 0 pinrrles T P N H aiid 1.0 pmolr GTP in place of C D P M

0.05 0.40
- . .....

(11 R . I k n a m u r ,

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I'atironnenii a n d C . G u n t z , Compl. uend., 246,

m n (19~8) (2) V. Ginsburg a n d H. N . Kirkman, THISJOURNAL 80, 3481 (1958). (3) Abbreviations: G D P M , guanosine diphosphate mannose; CTP, guanosine triphosphate; T P N H , reduced triphosphopyridine nucleotide; T P N , triphosphopyridine nucleotide; D P N H , reduced diphosphopyridine nucleotide. ( 4 ) Strain Ad5 ( A T C C 12657). (i) E. Cabib and 1,. 12. l,eloir, J. Riol. Chcm., 206, 779 (1954). (1;) 1'. hl. Kossal, Arrslruiian J. Ez#lZ. Bid., 31, 583 (1953). t i ) 2. Dische and I,. B. Shettles, J. B i d . Chcm., 175, 595 (1948).

Vol. 80

butanone-acetic acid-saturated boric acid solution butanol-acetic a ~ i d - w a t e r ,phenol-waterg ~ or pyridine-ethyl acetate-water.'O These solvents readily distinguish fucose from rhamnose. Further evidence for the identity of this sugar was indicated by the fact that it was active as a substrate for Lfucose isomerase." The reaction product, presumably L-fuculose, was detected by means of the cysteine-carbazole reaction .I1*l 2 The requirement for T P N H is shown in Table I . It is evident from structural considerations that the formation of the t-fucose derivative is a complex reaction which probably involves several steps. The nature of these steps remains to be elucidated. (8) W. R . Reis a n d T. Reynolds, Xature, 181, 7R8 (1958). (9) S. M. Partridge, Biochem. J . , 42, 238 (1918). (10) hl. A. Jermyn a n d F. A, Isherwood, .'bid.,44, 40)2 ( l Y i Q ) . (11) hl. Green a n d S. S. Cohen, J . Bioi. Ckem., 219, 5.57 (1950). (12) Z. Dische a n d E. Borenfreund, i b i d . , 192, 583 (1951).

NATIOSALIxs.rIn-,mOF & M E T A B O L I C r)ISEASES NATIONAL IXSTITUTES OF HEALTH U. S. PUBLIC HEALTH SERVICE RETKESDA,MARYLATII ARTHRITIS

\'. GINSBURG

RECEIVED ]iri.y 7 , 103S ~-

CRYSTALLOGRAPHIC EVIDENCE FOR THE RELATIVE CONFIGURATION O F NATURALLY 0CCURRING ISOCITRIC ACID'

Receiitlv Creelistein and his c o - t ~ o r k e r shave ~ studied the stereochemistry of the isocitric acids and alloisocitric acids and hdve concluded that the configuration of the a-carbon atom in the naturally occurring isocitric acid is I , ~ . Gawron and Glaide3 on the basis o f pk' measurements, have concluded that in the isocitric acid lactone the two carboxyl groups are cis with respect to the $-lactone ring while in that of alloisocitric acid the two carboxyls are trans. Thus. if the a carbon is in the L configuration, the formula in the Fischer convention for the naturally occurring isocitric acid is I. COO!I HOCH

I

Irc c o o ~ i

pP COOH 1

Through the kindness of Dr. H. B. Vickcry and Dr. D. G. Wilson of the Connecticut Agricultural Experiment Station we have been able to carry out an X-ray structure analysis on excellent crystals of the monopotassium and rnonorubidium salts of the lactone prepared by them from the isocitric acid occurring in the leaves of Bryophylliim calycinzim. These salts are isomorphous on the orthorhombic (1) Supported b y a grant (Cl?53) from t h e National Cancer Institute, Public Health Service. (2) (a) J. P. Greenstein, N. Izumiya, M. Winitz and S . &I. Birnbaum, THISJOURNAL, 77, 707 (195.5); (b) M. Winitz, S. M. Hirubaum and J. P. Greenstein, i b i d . , 71, 716 (1955). (3) 0. Gawron a n d A . J. Glaid 111. i b i d . , 1 7 , 6638 (195.5).