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Mickle disintegration of S. faecalis cells in 0.1 h? phosphate buffer of PH 7.2 containing 0.05 hf magnesium chloride. The reaction mixtures were prepared by dissolving 20 micromoles of thymidine triphosphate and 30 micromoles of a-D-glucose-1phosphate in 0.5 ml. of enzyme solution and incubating a t room temperature for 1and 5 hour periods. Examination of these samples by paper chromatography in a solvent system of ethyl alcohol and 1M ammonium acetate of pH 7.5 (7 : 3 by volume) revealed the presence of three new ultraviolet absorbing compounds with Rf values of 0.33, 0.25 and 0.20. A comparison of these values with those for reference compounds in Table I indicates that the compounds with Rf values of 0.25 and 0.20 are probably thymidine monophosphate and thymidine diphosphate, respectively. The compound a t Rf value of 0.35 yielded ultraviolet spectra typical of thymidine derivatives and on acid hydrolysis was converted to thymidine monophosphate, inorganic phosphate and reducing sugars. When thymidine triphosphate was incubated with the enzyme extract in the absence of glucose-1-phosphate, small amounts of thymidine monophosphate and thymidine diphosphate were produced but the compound with R f value of 0.35 was not produced in this reaction mixture.
‘I
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Hog kidney amidase, activated by magnesium ions,6 hydrolyzed the racemic amide 50% after 24 hours, 7570 after 4 days a t 38’ and addition of fresh enzyme on the third day. Preparations of D5’ the L-acid showing rotations as high as [ c Y ] ~ ~38. were 97y0optically pure as assayed by D-amino acid oxidase.’ This leads to a calculated value of [prl20~ -400’ for pure 111, Le., close to five times that of L-proline, [ff]*’D -86°.8 Yields of l O O % of I11 were not obtained because of the formation of gummy side products during the long reaction time. All analyses were satisfactory. Acknowledgment.-%’e are greatly indebted to Dr. S. M. Birnbaum for his valuable assistance and cooperation in the enzymatic resolution and assays.
TABLEI THE Rt VALUESOF REFERENCE COMPOUNDS IN SOLVENT M AMMONIUM ACETATE OF SYSTEMOF ETHYLALCOHOL-1 PH 7.5 (7:3 BY X’OLUME)
-
Compound
Thymidine triphosphate Thymidine diphosphate Thymidine monophosphate Thymidine Thymine Uridine diphosphate glucose Uridine triphosphate a-n-Glucose-1-phosphate D-Glucose L-Rhamnose
( 6 ) S. M . Birnbaum, “Methods in Enzymology,” Academic Press, Inc., New York, N. Y . , 1955, Vol. 11, p. 397. (7) L-Amino acid oxidase from snake venom converts 111 quantitatively t o pyrrole-2-carboxylic acid, a reaction which bas been used for a uew spectrophotometric assay: H. H. Weissbach, A. V. Robertson, R. Witkop and S. Udenfriend, Ami. Biochcm., in press. (8) D. Hamer and J. P. Greenstein. J . Biol. Chem., 193, 81 (1951).
Rf
value
0.11 .20 .26
.i6
. ‘io .22 .07 .18 .60 .68
The ultraviolet absorbing material a t Rf value of 0.35 was isolated by a paper chromatographic method in yields approximating 60% of the original thymidine triphosphate. Hydrolysis in 0.02 N hydrochloric acid for 1 hour a t 100’ converted the XATIONAL INSTITUTE OF ARTHRITIS AND hIETABOLIC DISEASES A. V. ROBERTSONproduct from the 1-hour incubation period to thyNATIONAL INSTITUTES OF HEALTH B. WITKOP midine monophosphate, inorganic phosphate and RETHESDA,MD. D-glucose and the product from the 5-hour sample RECEIVED AUGUST12, 1960 to thymidine monophosphate, inorganic phosphate, D-glucose and L-rhamnose. Quantitative values for these hydrolytic products were obtained by ultraA ROLE FOR THYMIDINE NUCLEOTIDES IN violet absorption for thymidine monophosphate, THE BIOSYNTHESIS OF L-RHAMNOSE1 by a modified Fiske-SubbaRow method for inorSiY: ganic phosphate2 and by the cysteine-sulfuric acid The synthesis of thymidine diphosphate glucose reaction for reducing sugars.a Molar ratios of and its conversion to thymidine diphosphate rham- these products are recorded in Table 11. The presnose has been observed in reaction mixtures of ence of D-glucose and L-rhamnose in the acid hythymidine triphosphate] a-D-glucose-1-phosphate drolysates was confirmed by Rf values in three difand a n enzyme preparation from Streptococcus ferent solvent systems, by enzymatic assay with faecalis. The enzyme extract was obtained by C. H. Fiske and Y.SubbaRow, J . Biol. Chem., 66,375 (192.5). (3) 2. Discbe, “Methods of Biochemical Analysis,” Vol. 11, edited b y D. Glick, Interscience Publisbers, New York, N. Y..1955, pp. 3 13-358. (2)
(1) Published with the approval of the Director as Paper No. 1047 Jnurnal Series, Nebraska Agricultural Experiment Station. ported in part by a grant from the National Science Foundation.
Sup-
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glucose oxidase4 and by the characteristic absorption spectra of the reaction product of L-rhamnose in the cysteine-sulfuric acid test.5 These results indicate that the product from the 1-hour incubation mixture is essentially pure thymidine diphosphate glucose and the product from the %hour incubation is apparently a mixture of thymidine diphosphate glucose and thymidine diphosphate rhamnose.
This reaction sequence is consistent with the observation that specifically labeled glucose is converted to L-rhamnose without randomization of the label6 and probably accounts for the occurrence of thymidine diphosphate rhamnose in several strains of b a ~ t e r i a . ~In ~ ~this . ~ scheme, reaction 1 illustrates the formation of glucose-1-phosphate (G-1-P) from glucose (G) and adenosine triphosphate (ATP) via hexokinase to yield glucose-6-phosphate (G-6-P) and via mutase to yield G-1-P. Reaction TABLE I1 2 indicates the synthesis of thymidine diphosphate MOLAR RATIOS OF HYDROLYTIC PRODVCTS FROM THYNIDINE glucose (TDPG) by a mechanism similar to that for DIPHOSPH.4TE HEXOSE COMPOUSDS FROM T H E 1- AND 5-HR. the synthesis of uridine diphosphate glucose. The REACTION MIXTURES enzyme which catalyzes this reaction may be apThymidine monophosInorganic Total propriately termed a thymidine diphosphate gluHr. phate phosphate hexose Rhamnose cose pyrophosphorylase. Reaction 3 is a diagram1 1.00 0.94 1.05 0.06 matic representation for a series of reactions result5 1.00 0.93 0.96 0.28 ing in the conversion of the glucosyl moiety of That the D-glucosylunit of thymidine diphosphate TDPG to the rhamnosyl moiety of thymidine diglucose is converted to the rhamnosyl unit has been phosphate rhamnose (TDPRh). These reactions established by the results shown in Table 111. In result in epimerizations a t positions 3, 4, and 3 and these experiments, samples of 0.8 micromole of reduction a t position 6 of the glucosyl moiety of thymidine diphosphate glucose in 0.05 ml. of enzyme TDPG. Reduced triphosphopyridine nucleotide extract were incubated for 5 hours with 0.15 or 0.3 (TPNH) is required for this series of reactions and micromole of the compounds listed in the table. in the crude reaction mixture is probably supplied The thymidine diphosphate hexose fraction from by other nietabolic reactions occurring in the syseach reaction mixture was separated chromato- tem. The thymidine diphosphate rhamnose probgraphically and L-rhamnose and D-glucose in the ably participates in transglycosylation reactions fractions were determined quantitatively. The as a donor of rharnnosyl units in the biosynthesis of values in Table I11 show that the disappearance of heteropolysaccharides. (6) W. H . Southard, J. A. Hayashi and S. S. Barkulis, J . B a c k r i o l , , glucose was accompanied by a stoichiometric iti78, 79 (1959) crease in L-rhamnose in the reaction mixture. (7) J. L. Strominger and S. S. Scott, Biochim. Biophys. A c f a , 36, TABLE I11 MICROMOLES OF L-RHAYXOSE AXL) D-GLUCOSE rs m E HYDROLYS.4TES O F THYMIDINE DIPHOSPHATEI I E X O S E FRACTIOXS PREPARED AS DESCRIBED IN THE TEXT
Compound added
Sone TPNH (0.15 p M . ) T P S H (0.3 &I.) D P K H (0.3 p M . ) Dialyzedb TPXH (0.3 p h f . ) Dialyzed + D P S H (0.3 p M . ) Glutathione (0.3
+
@moles of Lrhamnose
pmoles
0.03 .l7
0.79
of
D-
glucose
Conversion t o 1.-rhamnose," CI
.18
.?I8 64
.11
,TI
0 17 26 19 11
.01
. 79
I
,05
. 73
:i
.21
.64
pM.)
G-1-P (0.3 p M . ) .03 78 I) The figures have been corrected for the sninll amount of 1.-rhamnose initially detected in the TDPG preparation by the colorimetric procedure employed. The enzyme extract was dialyzed for 24 hours in water and for 3 hours in 0.1 AI phosphate buffer containing 0.05 JI magnesium chloride. a
552 (1959). (8) R. Okazaki, Biochein. Biophys. Res. Comnt., 1, 34 (1959). (9) J. Baddiley and S. L. Blumson, Biochim. Biophys. A d a , 39, 376 (1800). (10) Dnw Chemical Co. l ~ e l l ~ ~University w, of Nebraska, 19591960.
DEPARTXENT O F BIOCHEMISTRY AND N U T X I T I O N JOHN H. PAZUR UNIVERSITY O F NEBRASK.4 LINCOLX, NEBRASKA ELDON W. S H U E Y I O RECEIVED JUNE 27, 1980 METAL-HYDROGEN BONDING I N METALLOCENE COMPOUNDS'
Si?.: Evidence provided by our infrared spectral studies on a wide range of ferrocene and some related metallocene derivatives has established a revealing example of the active electronic role which can be exercised by the metal atom in appropriate reactions of these metallocene systems. Study of the hydrogen bonding characteristics of ferrocene atnd related derivatives has shown the metal atom to be the site of a substantially strong intrarnolecular hydrogen bond involving the electrons of the metal atom acting as proton acceptor.
On the basis of these findings, a reaction sequence suggested for the conversion of D-glucose to L-rhamnose by the enzyme system of S. faecalis is o u t lined in the accompanying equations
+
G A T P ---+ G-6-P +G-1-P TTP f G-1-P -3 T D P G P-P TDPG -4- TPNH --+ TDPRh TPS+
~.___
+ +
(1) (2) (3)
(4) L . M. White and G. E. Secor, Scienre, U S , 495 (1957) (.5) 2 . Dische a n d I.. R . Shrttles, ./, Riol. Thrni , 175, 595 ( 1 948).
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(1) This research was supported jointly by the Army, Navy anti Air Fnrre u n d p r Signal C.orps contract DA-36 039s~-78105.
