A study of carbamyl phosphate synthesis in Streptococcus lactis

A Study

of Carbamyl

Phosphate

Synthesis

in Streptococcus

lactis

properties of the enzyme wre dctwmincti ISTRODUCTIOS

Hiotin lm been shown t’o lmvc an indiwet role in tlic qnt~licsie of ornithinc transcnrbainylase in StYeptocol‘clcs lnctis 8039, alid tlie possibility that a deficiency of the >ubstrate, carbamyl pl~oqhate, might demm: the qnthesis of ornitliiae tmnscarI~amyla~e x-ns coneiderctl (I). Uiotin dcficiency does cause a loss in the ability of rat liver l~oniogcnatc~ to convert ornithinc, I)icarbonatc, and amnonium ion to citrullint, and supplenmts of X-cahanlyl glutnlllntc hut not of biotin restore tile artivity 121. It has Gco heen sliom that the A*carhiyl glrltamat~e rcquiwiuent i:: conccmcd wit11 the syntlmia of carbnmyl phos]‘lrate and not with tllc tr;ln~carbam~lation ixB:iction ( 3’) Since mppleincnts of S-carbamyl glutamate arc not required for t,llc yynthc& of rarbam;r-1 pllospliatc in rel)ort’cd bnctci+al prcpm~tions (4 1, a hdy of cnhm~l ~~ho~pl~at~cqxtlwh in S. lnctis I~:16 undcrtakcii to dcknninc whether or not hiotin ha* :I lxirticular role in the fiyntllrsis or funct,ioning of this rnzyme. The lack of an c+Ycc.t,of a l)iotin deficiency upon production of th mzymc euggmts that hiotin is not involwd dirertlg in the production. The dccrrnsctl hiotin content of active fractions of the cnzyinc~ after each step in purification

and tlic inabilit~y of akliii to affect the activit,y of the lwificd cnzyirle Suggest that Giotiri is not a cofactor in the enzyme. Of lmrticular interest, liowevcr, is the olwrwtioii tliat sulfate ion specifically protects tlw VIIZ~IIIC and aho reactivates ~~rlZylllc

~JIY~):iratlon.c:~~~IOIis

Ivhich

halve

beer1

iii-

activatccl t)y di:tlyC, hat’ treatment’, or aging.’ 8om of tllc Iwopcrtiee of tlic purifictl mxyme fraction were nlso determined.

1 IETIIOTE Protcin (8): odthine tr:lnscnrhnni~lnsc (,I), anal the biotin content of enzyme fractions (1, 9) wew clrtcrminetl 1)y prc\-iously descril)ed metboIls. __- - ~-- ~_~ -~ __.-.1-1 lxcliminxy communication on the purific:~-

525

5%(i

RAI’EL,

IICMI’HHETS

Carbnmate kinaee activity was dclcrmined routinely in 1 ml. solution which conttained ATI-‘, 8 mngnesittm chloride, 10 ptnolcs; Tris pmoles; buffer, 50 ~mol~s; nt,-ornithine n~ottohytlrochlocnrl,:lt~lnte, 250 ride, 20 pmoles; ammonium Cranscnrhamyl:~se, 10 Kg. ptnoles ; and ornithine llrotein (specific activity, 77.000). A solution of the first four ingredients adju;;tcd to pH 8.7 MW l)rrpared at 2.5 times the final concentration and stored at -20”. Just prior to testing, orttit,hine Ir;lnsc:lrb:lm?-1:lre and solid animonirlm cnrbamnte in the approprintc amounts were added to the soluiion of the firsi- four ingredirnts at 4”, and 0.4 ml. of the reslllting solution was added to cxrh tube containing a rate-limiting amount of carbatnate kinasc in 0.6 ml. water. ;\Et~r 5 min.’ incuhation at 3S”, Ihc reaction was stopped by the adtlition of 1 ml. of the sttlfuric-phosphoric acid mixture used in the citrttlline c.olor assay. Citrulline was determined calorimetrically (10) on npproprinte aliquot s. Specific activity is expressed as micromoles of cnrbampl llhosphnte synthesized (as measltred by the amount of ~itrulline synthcsized) per milligtxm of protein per hour. Other modifications of the assay proccdurc used in VWkin cases nrr indicated in the tablrs and figures. In sonic experiments in which only the k&se enzyme was used. c~~rbamyl l)hosphnte was tlct~rmined by the addition of an cclunl \,olume of 2 N sodium hydrosidc, allowing the rcnction system to stand for 10 min. at. rootn Ccmperatltrc, and measuring the Pi libctxtcd by the Fiske and SubbaRow tnethotl (11, 12). PRODVCTI~IY MD PURIWXTIO~; CARRUMTE I
OF

A rulture of S!~cptococclcs I0cfi.s SO39 grown from StOCli culture in 10 ml. of n previously described inoculum medium (13) for 16 hr. at 30” was added to 1 1. of a medium dcscribcd I)y Knivett (14). The medium was tnotlificd by reducing the nrginine concentration t,o 0.75% and by nddition of 1’6 ammonium sulfntc and 0.1% sodium bicarbonate (solid added to the c~oolcd, sicrilc medium just prior to inoculation). After 12 hr. growth at 30”, the 1 1. culture was then used to inoculate 10 1. of t,he s:unc medium. After 12 hr. incubation at 30”, the &Is were harvested by means of a Sharples centrifuge, ~~shctl with 0.85% sodium chloride. and resuspended in 200 ml, of 0.04 ‘$1 Tris buffer. pH 8.5. The cells \vvcrc csposcd in 50-ml. portions to sonic oscillation in a Rnythron lo-kc. oscillator for 30 min. The suspension of disruptcd cells was ccntrifugcd at 20,000 X g for 1 hr. at 4” , and the cell tlebris was discarded. The ctlllfree estx~act. containing :~pprnrimntrlq 5 g. protein,

.lND

SHIVE

~vas diluted with Tris buffer to a concrntration of 20 my. protcin/tnl. Ml subsequent procedures were carried out at 4”; the precipitnl cs wet’c collcctctl by ccntrifugntion at 20,000 X 9 for 20 min.; and all fractions were stored at -20” in at least 0.5 M ammonium sulfate. Cold saturated nmtnonium sulfate, 225 ml., l)H 7.4, MU added alo~ly with st,irring to 225 ml. of cell-free extract. After 30 nrin. the pl,clcipiinte was rcmo\-cd by ccntrifugntion. The precipitate containc~cI only lo%, of the cnrb:nn:~te kinuse act ivit) and was discarded. To the 50% saturated supcrnatant solution, an additional 225 ml. of cold, sxturai cd nmmonitml sulfate solution, pH 7.4, was :tddcd with stirring. The prt,c*ipitatc kvas collected by ccntrifugation and resuspended in 0.04 M Tris buffer, pH 8.5. This fraction, containing the major portion of the carbntnate kinase activity, was dialyzed against 0.04 IV Tris buffer. pH 8.5, for 2 hr. at 4” and diluted to a protein concentration of 20 mg./ml. ,\ second ammonium sulfate fractionation was carried out in the manner described abo\,e. The second 50-66% saturated ;m~tnonium sulfate ftxct,ion was dialyzed nt 4” against 0.015 Al glycinc, pH 8, for 2-4 hr. and then placed on a 10-g. DIME-cellulose colutnn which was 2.5 cm. in diameter and 29 cam. in length am1 which had bcc,tt ec~uilibraled with 0.015 N glyck, pH 8, at 4”. The column was washed with 250 ml. of 0.015 JI glycine, followed by 250~ml. portions of miutures of 0.015 JI glgvine and 0.02, 0.04, 0.06, and 0.08 N phosphntc at a pH of 8. The c*arlxunnte Iiinase activity xx9 rcmovccl with a tnixtlu,e of 0.015 111 glycine and 0.1 121 phosphntc. pH 8. Solid ammonium sulfate was added to the active fractions to bring the per cent of saturation to SO. The l)rccipitntc x-:as collected by centrifugntion and dissolved in 0.04 ill Tris, pH S.5.

The cell-free cxtrnct obtninrd as describetl above was exceedingly rich in ornithine transcnrbamylase (specific activity, 6000). The ratio of specific activities of ornitliine tranacarhxmglase to carbatnyl phosphate synthctnec was approsimatel!, 175: 1. The two ammoniun~ sulfatr frnctionntions retluccd t,hc ornithine tr~~nscnrbamylnse content to 20% of the rrutle cstract. On ihe DEAE-celllllose column most of the ornithine trans~nrbnmyl:IW was rcmovld by the 0.015 ;1I glycine and 0.08 lf ~~hosphate tnixtltrc. Tho nctivc carbnmate Iiimse friKti0nS (0.1 111 phoSphatei contained 0nlJ 0.5% of the ornithine tr:lttsc:lr),:lm?;lnsc prrsent in the crude extract. The major portion of the olnithinc trnnsrarbatnylnse had remained in the 66% saturated am-

CARBAMTL

I’HOSPILATE

YTNTHESIS

,527

528

RAVEL,

HUMPHREYS

SND

SHIVE

trat’ion of sulfate ion to which the enzyme is exposed and not upon the concentration of the sulfate ion present during the enzymic conversion of carbamate to carbamyl phosphate. The specific activity of an enzyme prepration which has been inactivated by dialysis can be restored to approximately 607; of the original activity of the preparation bcfort dialysis by treatment with sulfate ion. The effects of time and tempcruturc upon this reactivation of carbamate kinase by sulfate ion arc shox~~ in Fig. 1. The rate of renctivut’ion by sulfate ion increases al,proximately threefold with each 10” increase in temperature. This apparent enFIG. 1. The rffecis of time and tcmperatuw on crgy of activation strongly indicates that the reactivation of carbnmatt kinase by sulfate a chemical reaction involving appreciable ion. A purified preparation of carbnmate kinase (fraction IV, specific> activity, 1750) was dialyzed bon(l energies is involved. Sulfate ion could for 7 hr. against 0.04 JI Tris buffer, pH 8.5. The be directly incorporated into the protein inactivated preparation was t,hen diluted with an but may only initiate or catalyze the transeclunl volume of 2 dl nmmonium sulfate, incubatctl forniation without direct mcorporation. at the times and temprratuws indicated ahore, Since it would be unlikely for such high for ra~hnnx~tc kinwc activity. and assaved . concentrations of sulfate ion t’o exist under physiological conditions, it would be of inof the transcarbamylases and can be re- tcrcst to determine the effect of the sulfate placed by a pept’ide of aspartic acid. donor, 3’-phosphoadenosine 5’-phosphosul&is indicated in a preliminary communifate on the reactivation of carbamnte cation (5), sulfate has a role not only in kinase, particularly if sulfat’e itself is instabilizing carbamate kinase but also in re- corporated into the enzyme. activating preparations which have been Some additional determinations on the inactivated by aging, dialysis, or heat treatproperties of preparations purified some 50mcnt In the absence of sulfate ion, there is fold with spwific activities in the order of 2000 wcw niadc~.The optimum l&I for tlic wa 50:; loss in activity on storage at -20” for 1 week, an 80% loss on dialysis overaction is 8.9-9.1 and tlic optimum temperxnight at 4” against 0.04 M Tris buffer, pH turc is 37-39”. The rate of the reaction is 2.8 8.5, and an 857; loss on heating at 60” for tinw as fast at 35” as at 25”. The bivnlent 2 min. This loss in activity is prevented if cat’ion requirement can be satisfied cqunlly these trentmwts arc carried out in 0.5 ~1.1 well by 0.01 JL Mg++ and to a lesser extent ammoniuin sulfntcx. When carbamate kinnse by 0.01 A1 Fcif (activity, 507; of control) is inactivated as described above, the en- ant1 0.01 X Co++ (activity, 10% of conzymic a&vi@ ran bc largely restored by trol) ; Cd++, Cu++, and Pb++ are inhibiallowing the inactivated preparation to tory cvcn in the presence of magnesium ions. stand in ammonium sulfate solution for 1 The ATP cannot bc replaced by the triphosphates of guanosinc, inosine, uriclinc, hr. at, 0”. Both sodium sulfate and potnssium sulfate arc as effective as ammonium or cytidine. As calculated from Linen-eaverenzymic activity, Burk plots, the K,,, values are as follows: sulfate in restoring Mg+ f, 2.2 x 1O-3 A!; &In++, 2.7 x lo-” ~hercns ammonium carbonate, ammonium chloride, potassimn l’hosphate, and the X; and ATP. 3.4 X lo-” M. These values residual ash from heating ammonium sul- are dcpcndent’ upon t’he spwific concentrnfntc arc without effect, under the same con- tions of metal ion and ATP. ditions. The dcgrce to which the enzqnc is Sodium carbonate-bicarbonate and nmrcactiyntcd is dependent upon the concennioniuin sulfate replace ammonium car-

CARBAMYL

PHOSPHATE

.i'1!)

SYNTHESIS

banlate in the assay procedure hit are Ics:s c~ffectire, :ts indicated in Fig. 2. By the time tlicl cnhmatc has hem tlissolwd iii tlic wuction mixture and plwctl in tlic tubes for incubation, the carbainatc concentration is only 50-605, of that :tddcd. Carlmlnatc wt.‘: deterinined by a inodific:~tion of a procrrlu~~c reported for cyanatc ( 17). The ainnionium-ion concentration was tlc~terii~iiicd Iy pipctting an appropriutc aliquot of the tv:lct#ion mixture directly into Ncsaler’:: rc:qynt and another aliquot into 0.1 S sulfuric acid before the addition to Kessler’s rcagcnt. The diffcrcnce twtween the nulrlloniuln ion directly detcmllincd and aftcrr :Icitl t~reatincnt is a measure of the carhtu:hte-ion concentration. At the tempemtrlrc of the incubation (38” 1i the concenI

I

I

I

5

IO

15

20

PS AMMONIUM

l

o

J/

CARBAMATE

SODIUM CARBONATE-BICARBONATE AND AMMONIUM SULPHATE

!

-

NOT

--

PREINCUBATED 5 MINUTES BEFORE ADDITION OF ENZYMES

PREINCUBATED

I

25

ENZYME

FIG. 3. The effect of coupling the cnrhnmylation of ornithine with t,he synthesis of carbnmyl phoppIrate. f’~o./z A: The reaction system containe(l in :L volume of 1 ml.: ATP. 8 pmoles; MgCL , 10 ~moles ; :Imnioniutn cnrbLnn:lte, 250 ,kmoles : 11Lornithine, 20 fitmoles; ornithine trnnscnrb:lm?ln~f,~ 10 pg. protein; and Tris buffer, 50 pmoles; pH S.9. The amount of carbnmyl pho.y)hnte syntheziwtl was tletermined ns the amount of citrulline formr~cl. C~rule B: Ornithine anti ornithine trnnscarbnm:.lride were omittcd from thv rcnction system, :i11t I 1hr c~rrl~;rniyl l)hoslth:tte ww measrved as the I’, libcr:~tecl by clilutt :illinli. Both xnction sxwrnl: lucre incubntcd fol, 5 min. :rt 3s’.

530

RAVEL.

HTMPHREYS

AND

SHIVE

phate occurs more rapidly with carbamate, these results do not preclude the possibility that carbonate may be utilized directly with ammonia by the enzyme. Carbamyl phosATP formed Carbamate kinase phate consumed The omission of ornithine and its transcarbamylase in the assay for the kinaee pnmles pmoles pg. pvotein decreases the yield of product as indicated 2.2 2.5 10 in Fig. 3. If the enzyme concentration is ina .7 3.6 20 creased to 200 pg. without being coupled n The re:tct,ion systenl containing AJ>P, 10 to the carbamylation of ornithine, the car~moles; carbamyl phosphat,e, 10 pmoles; MgCln , bamyl phosphate produced is increased to 10 ,~~molrs; and Tris buffer, 50 pmoles; pH 8.9, in a 4.0 pmoles, :L twofol(l increase in product total volume of 1 ml. w:ts incubated at 38” for 5 for a tenfold incre:w in enzyme concentramin. The c~arha~nyl phosphate U-RS me:wured :ts tion. the l’i lil)er:tted by dilate alkali, and the ATI’ The synthesis of curbanlyl l~hoaphate is formed w:ts tlcterminetf by R modification of the reversible. as indicated in Table IV. and an lucifernsc :~ss:ty (18). cquivalcnt alnount of ATP is produced frown ADP for the alnount of cnrbamyl phoq’liatc consumed. A wpl)lcinent of amnionium carbamate npprcci:abl\- decreases the rntc of the rewrsc reaction, as indicated in Fig. 4. If the cnzymc conwntration is inci~cascxl to 200 pg. in tlic prcwncc of nniinoniulii carbamatc and the ammonium salt of c’alballl~l ~,llosplm ~pTpwl flm~ th! tlilitlliulu salt by pnss;:qc through :t T)ow(~): 50 column (NH,+ forni 1 at 4”l i:: wed, the anwunt of carlxunyl phoq~hwtc consumed is 4.5 pniolcs. Thus, tlrc rcverw rwvtion and the forward rcwtion under iclcntical contlitions prowctl at abollt qua1 rates in t,lic ~nxwncc of a high concentration of ainmonium carbnn~atc; but in tl1~1nlxwnrc of this ~ulq~lcmcnt, the formation of .1TP prowctls more rapidly than the formation 0 5 IO 15 20 25 30 35 of c~arbnniyl pliosphate. as has been pw,ug ENZYME Co1lhly rq)ortctl (11 1. FIG. 4. The effect of ammonium cnrbamnt(> on the phosphorylntion of AD1 by ~nrbamate kinnsc. CI(I.LY A : The reaction system eont,nined in a YOIrime of 1 ml.: .kDP, 10 pmolea ; MgCL , 10 j~moles; cnrhnmyl phosphate, 10 ymoles; :mtl Tris buffer, 50 pmoles; pH S.9. CURLY B: The reaction system c,ont.:iined, in adtlit~ion to the above, 250 pmoles ammonium carl~nm:rtc. Both reaction systems were incltbated for 5 min. at 38”, and the c:nbamyl phosphate vas n~c:~surcd as the Pi libcrntcd by clilutc nlknli.

So significant’ diffewncc in the cffccts of tcmperat~urv, pFI, metal-ion requirement, or ATP rcquiremcnt could 1~ demonstrated using ammonium nn(l carbonate salts as the substrntc in lieu of ammonium carbamate. _~lt,llougli tbc a\-nthwir of c~arbamyl l)ho~-

2. F~:r.tm~, (;., ..,ND I,.ww. 192,447 ( 1951).

H. A\ ., J. Ij-;iol. Clwul.