Oct. 20, 1'352
5157
THECYTOSINE DEAMINASE OF YEAST
which had the properties of "a-sphingosinc ~ u l f a t e , "i.e., ~ it graduallv became yellow on exposure to air, and upon irradiation with ultraviolet light (Hanovid utility lamp) specimens exhibited a whitish fluorescence whose intensity considerably exceeded that of a sample of quinine sulfate (violet fluorescence) which was examined under comparable conditions. .4na2. Calcd. for (ClsH370~X)z.H2S04: C, 62.0; H , 10.99; S,4.02. Found: C, 61.4; H, 10.95; S , 3.83. A sample of sphingosine, freshly prepared oia the acid hydrolysis of beef cord, was kindly supplied by Dr. Irving Zabin, Physiol. Chem. Dept., the Medical School, V.C.L.A., Los Angeles, Calif. The sample, recrystallized from acetone, had m.p. 96".
Triacety1sphingosine.-This compound was prepared from sphingosine sulfate according to the directions of Carter and co-workers24 and purified by recrystallization from acetone. The material thus obtained had [a]27D -10.9' (c 1.8, chloroform), m.p. 101-102° ( r e ~ d [.a~I z ~ 5 D -11.7' (chloroform), m.p. 101-102'). Anal. Calcd. for C?aHanOjS: C, 67.7; H, 10.18; S , 3.29. Found: C, 67.5; H, 10.25; N, 3.02.
Acknowledgment.-A grant-in-aid from Research Corporation is gratefully acknowledged. The author also takes pleasure in thanking Dr. David R. Howton for stimulating discussions. NEWYORK53, S. Y .
[CONTRIBUTION FROM THE DEPARTMENT O F BIOCHEMISTRY, COLLEGE O F PHYSICIANS
AND
SURGEONS, COLUMBIA UNIVERSITY]
On the Cytosine Deaminase of Yeast1 BY JACOB KREAM AND ERWINCHARGAFF RECEIVED MARCH22, 1952 The description of the isolation of partially purified, stable cytosine deaminase preparations from bakers' yeast is followed by a study of the properties of the enzyme. I t degrades cytosine with the production of equimolar quantities of uracil and ammonia in a unimolecular reaction at a p H optimum of 6.9. The effects of enzyme and substrate concentration and of temperature on the course of the enzymatic action have been investigated. The Michaelis constant has been found as 8.4 X 10-8 M , the temperature velocity constant as 19,500 cal./mole. Of many other pyrimidines studied, only 5-methylcytosine was converted to thymine by the enzyme preparations. Isocytosine acted as an inhibitor. Conclusions as to the specificity requirements of cytosine deaminase are discussed, and a procedure for the chromatographic separation of 5-methylcytosine from other pyrimidines and for its quantitative determination in minute amounts is described.
The present paper considers the cytosine de- deaminases and the discovery that yeast was a good aminase of yeast in some detail. Brief accounts of source for the isolation of cell-free preparations of deaminase2j12prompted a more detailed some of the phases of the work have a ~ p e a r e d . ~ cytosine .~ Enzyme systems capable of deaminating cytosine investigation of this enzyme, also with respect to its to uracil have been encountered rather infre- specificity characteristics. quently. Extracts of animal tissues appear in Experimental general to be unable to degrade cytosine itself, Material.-The following numbering system will be used although the ribonucleoside cytidine is a t t a ~ k e d . ~ , ~ That dietary cytosine can be converted to uracil in describing the pyrimidines. 1 S---CHO in the body has been both denied6 and affirmed.' I1 I1 Indications of the production of ammonia from 2 HC CH 5 cytosine, incubated with fowl blood, have been I 1 reported.8 3 S=CH 4 The presence in bakers' yeast of enzymes able to Cracil either was used as a purified commercial preparation deaminate cytosine and 3-methylcytosine to uracil or synthesized here.I4 Cytosine was prepared from uracil,'j and thymine, respectively, is well e s t a b l i ~ h e d . ~ ~as~ ~was ~ , ' 2-methoxy-6-aminopyrimidine. ~ 5-Methylcytosine A cytosine deaminase also has been found in E . and 2-mercapto-6-aminopyrimidinewere obtained through the courtesy of Dr. G. H. Hitchings of the Wellcome Recoli2,11j12and obtained as a cell-free extract"; search Laboratories, Tuckahoe, S . Y. For 2,fi-diaminobut this property does not seem to be inherent in all pyrimidine, 2-hydroxy-4,6-diaminopyrimidineand isoguanstrains.I3 The development of convenient micro ine (2-hydroxy-6-aminopurine) we are highly. indebted to procedures for the study of purine and pyrimidine Dr. A . Bendich of the Sloan-Kettering Institute for Cancer (1) Supported by a research grant from the National Institutes of Health, United States Public Health Service. This report is based on a dissertation submitted by Jacob Kream in partial fulfillment of the requirement, for the degree of Doctor of Philosophy in the Faculty of Pure Science, Columbia University. ( 2 ) E. Chargaff and J . Kream, J . B i d C h i n . , 175, 998 (1948). ( 8 ) J . Kream and E. Chargaff, Fedevalioit PI'oc..9 , 192 (1950). (4) G. Schmidt, Z . pkysiol. Chein., 208, 185 (1932). ( 6 ) j. P. Greenstein, C . E. Carter, H. W. Chalkley and F. M. Leuthardt, J . Nall. Cancer Insl., 7, 9 (1946). (6) L. B. Mendel and V. C. Myers, A m . J . Physiol., 26, 77 (1910). ( 7 ) L. R. Cerecedo, J . B i d C ' h e m , 75, GO1 (1927). (8) E. J . Conway and R . Cooke, Biochem. J . , 33, 457 (1939). (9) A . Hahn and W. Lintzel, Z . Biol., 79, 179 (1923). (10) A. Hahn and W . IIaarmann, ibid., 86, 275 (1926).
Research, New York; for isocytosine (2-amino-6-hydroxypyrimidine) and 2,6-dihydroxy-5-aminopyrimidine to D r . M. Hoffer of Hoffmann-La Roche, Inc., Sutley, S.J.; for 4-hydroxycytosine to Dr. M. Engelman of this college. The preparation of cytidylic acid has been described pre\-i0usly.~6 Cytidine was a purified commercial preparation. Quantitative Determinations.-Cytosine and uracil were separated by filter paper chromatography and estimated by spectrophotometry, as described in a previous p ~ b l i c a t i o n . ' ~ Essentially similar procedures served for the separation of thymine from &methylcytosine and the quantitative determination of the latter. For the techniques used, the recently published procedures for the estimation of xanthine and hypoxanthine should also be consulted.12 The separa( 1 4 ) D . Davidson and 0 Baudisch, THIRJ O U R N A I , , 48, 2379 i192G). 115) C. E. Hilbert and .'l' H. Johuson, i6id.. 52, l l Z 2 ll!43lll. (11;) IEAMIXASE' lactam form; the introduction of a substituent in Tqtal Expyrimltent position 3 protects the substances from degradadine (if tion. The intactness of the CH group in position 4 Iacuha- S-hlethylcytosine acdetion remain1n.g Thymine formed counted aminaof cytosinc also seems to be essential: 4-hydroxyfor, tirin, microtime, micrucmoles "; minutes Y moles cytosine, .~-amiriocytosiiieor isoguanine are not A0 (j , 5 0 , f),? :i-1 2 (1.27 97 82 attacked. If >-methylcytosine and cytosine are 120 6.5 ,U,5 :34.!) 28 IO(I 85 treated as substrates by the same enzyme, position 5 of cytosine may lie considered as not essential for a In each assay, 1 nil. of it 0.2,5% aqueous enzyme ~0111the action of the deaminase. Finally, the amino tion and 1 mi. of a 3:3 m.lI solution of 5-methylcytosine in 0.4 JI phosphate buffer o f pI-1 fj.!J wcre iricuhated a t 37.T,'. group must bc situated in position (i: isocytosine is The values reported refer to O.02-mi. aliquots of t h e inis- not dcaininatetl by the yeast enzyme; it acts as an tures (corresponding to 0 . 3 micromole of initial s u h ~ t r ~ ~ t e l inhibitor. and represent averages of 3 clctcr~ninations. SEW YORK,N.Y. ,
,
Resistant and Inhibitory Compounds.--Sone of the othcr pyrimidines and analogs listed in the first section of the Experimental part was deaminated by the enzyme. 2-,4niino6-hydroxypyrimidine (isocytosine) and 2-rnercapto-8-aminopyrimidine depressed the rate of cytosine deamination. For instance, when ammonia production from 108 micromoles of cytosine by 2.5 mg. of enzyme in a total volume of 2 nil. of phosphate buffer (60 minutes, 3 7 . 5 " ) was studied
i 2 ( i ) Resting cells 01 E coli t h a t rxhil,it rytosine deaminase activity (re1 1 2 ) were. o n the other hand, lound to bring ahout the slow li1,crxtion of arnmotiia lrom isocytosine. ' ? i J I). i Ilrown. S a l u r r , 166, 1010 ( l S I . N j . 2 8 , 'I' I' \ I ' d n g a n d .T. 0 I.ampen, J . Bioi. ( l i r i i i , 194, i83 fI!l:21 I ? ! > > I'. I L)i Carlo, A S . Schults tind A 11 K e n t , i h i d . 194, 7'69 ! l!j.j2).
