Cysteine desulfhydrase activities of Salmonella typhimurium and Escherichia coli

I3:2

B I O C H I M I C A E F B I O P H Y S I C A AC'I

BBA 660~0

CYSTEINE D E S U L F H Y D R A S E A C T I V I T I E S OF SA LMO NE LLA

T Y P H I M U R I U M AND ESCHERICHIA ( O L I

GkBRIF.LGI'ARNI~R()b*

aXD M A N U E L

\

()Rlk(.k

D @ a ~ t e m e n t o de (,~ m tzca v 13wlo~ia C ~l~t/a~, C, nt~(, de lnz'~d~gacwn y de l:~l~td~os A van~ados del I P N , 3[dx~co, D F (.,lI)t~co)** (Recel\cd

A u g u s t 2 o t h , 1969)

SUMMARY

I Both wild type and a cysteme-auxotroph (cy~E) mutant of Salmonella (vph~mm'ntm LT2, as well as strains B and W ofEscher~chm coh, presented an reducible cysteme desulthydrase (I.-cystelne hydrogen sulfide-lya~e (deammatlng), EC 4 4 I I) actlwty free of catabohte repression The enzwnatm activity wab rapidly induced m the prebence of either glucose or glycerol 2 The cystelne desulfhydrase actlwty of S lyphzmurzl~m sho~ ed a pH optlmuln of 8 5 and, in intact cells, both cysteme and cystlne were used as ~ubstrate~, the presence of glucose did not enhance the activity Toluene-treated cells lost the abthty to desulfllydrate cystme, x~lule the actlwty towards cysteme wab lncrea~ed In contrast, the cystelne desulthydrase activity of E colz ~howed a pH o p n m u m of 7 2 and m intact cells the presence of glucose increased the activity Toluene-treated celN l ~ t all activity for the desulfllvdranon of cysteme 3 The enzymatic activity of S (vph~mur~im wab not inhibited ~n vztro by NH~+ unless they were added along with pyruvate Pyruvate alone was shghtly mlub~tory, wtnle H,,S had a strong inhibitory effect 4 The cysteme desulfl~ydrase activity ofS (~,phwu~r~um apparently ~b different from the cy~tathlona~e activity as both were reduced and reprebsed independently

INTRODUCTION

During the characterization of a cystelne-requmng nmtant of 5"almonclla (vph~murlum, it was flmnd that its growth m glucose minimal hqmd medmm wa~ hmlted by the rapid desulthydratlon of the required amino acid, a finding very ~mnlar to tho~e reported for cvstelne auxotrophs of Escherzchza cob 1,2 Tlus result and the fact that no lnfl~rmatlon about the cy~teme desulihydrase (L-C3.~teme hydrogen * Pre~ent addre~ C a h t , 9 4 7 2 o , U ~, A ** P o s t a l a d d r e s ~

13wthznl

t3tophvs

Department \partado

of Moleculal Bmlogy,

U n l v e r ~ l t 3 o f (. a h f o r n t a ,

P o s t a l ~4 74 °, M e x i c o 14, I) V , M 4 x l c o

4Lta 108 (107 o) 1 3 2 - I 4 2

13crkclc?,

(YsTEINE DESULFHYDRASE OF ~ typhzmurzum

133

bulfide-lyase (deamlnatlng), EC 4 4 I I) a c t i v i t y of S typhtmur~um was available p r o m p t e d us to s t u d y some of the characteristics of this a c t i v i t y , as well as some aspects r e l a t e d to its mducabxhty, a n d to c o m p a r e t h e m w~th those r e p o r t e d for the same e n z y m a t i c a c t i v i t y of E col¢-4,1~ MATERIALS AND METHOD%

Term t n ology The use given m this p a p e r to the t e r m cysteme desulfhydrase a c t i v i t y (L-cysteme h y d r o g e n sulfide-lyase (deammating), EC 4 4 I I) refers to the classical p s n d o x a l p h o s p h a t e cystelne deaulthydrase yielding HoS and the u n s t a b l e a - a m i n o acr-~ hc acid which bpontaneou~ly hydrolyzeb to N H 3 a n d P5 ruvlc acid (cf ref 4) Bactertal stratns Salmonella typh~muru~m LT2 was k i n d l y p r o v i d e d b y the late D r M Demerec M u t a n t strain SMIo3-2 wa~ o b t a i n e d from the LT2 p a r e n t a l s t r a t a as a s p o n t a n e o u s cystexne a u x o t r o p h resx~tant to s t r e p t o m y c i n (2o #g/ml) Escherzchm coh stratus B a n d W were k i n d l y provxded b y Dr Gene M Brown All strains were k e p t on nutrxent glucose agar slants, b u t for m u t a n t SMIo3-2 the m e d m m alao contained 4 ° # g / m l of L-cvstme Ch cm 1cals All chemicals used ~vere reagent were p r o d u c t s of S i g m a Chemical C o , from Cyclo Chemwal C o r p o r a t i o n All DL-lanthxonine and DL-cystathxonine were product~ of Dffco Laboratorle~

grade P y r l d o x a l p h o s p h a t e and amino acids except 0-acetyl-L-~erme t h a t wa~- p u r c h a s e d ainmo acids used were the L isomers, except B a c t o - t r y p t o n e a n d Bacto-Casamino acxds

Culture conditions The m m l m a l m e d i u m used x~as t h a t of DAvis AND MINGIOLI 5, cxtrate o m i t t e d The different c a r b c n ~ources were used at the concentrations Indicated m the t e x t Cysteine, cystlne, O-acetylserine a n d l a n t h l c n l n e were sterllxzed b y filtration and a d d e d to the corresponding m e d i a at the final c o n c e n t r a t i o n s l n d m a t e d m the t e x t Experiments of ~nduct,on and represszon of enzvmatzc actzv2tms The b a c t e r i a were grown overnight (i2 h) at 37 ° in the m e d i a and the culture conditions i n d i c a t e d for each e x p e r i m e n t Fresh m e d i a were m o c u l a t e d with a sample o f the cells grown o v e r m g h t (approx 5-1o ~_ of the final volume) a n d i n c u b a t e d u n d e r the a p p r o p r i a t e c o n d m o n s until the a b s o r b a n c e of the culture at 660 n m reached a value of o I (read m a Colemen J r s p e c t r o p h o t o m e t e r , model GA) A t t h a t time the cells were h a r v e s t e d a s e p t i c a l l y b y centrlfugatlon in the cold, washed twlce with sterile m m l m a l m e d m m and resuspended in a p p r o x 5 ml of the bterfle mmxmal m e d i u m Ahquot~ of this suspension were used to lnocculate the l n d u c h o n m e d i a W h e n the e x p e r i m e n t was designed to s t u d y the induction of the cysteine desulthydrase a c t i v i t y in a defined time, the m o c u l a t e d m e d m were i n c u b a t e d for t h a t time, a n d at the end of it the cells were h a r v e s t e d b y c e n t n f u g a t l o n in the cold, washed a n d uaed as ~uch or for p r e p a r a t i o n of t o l u e m z e d cell~ or cell-free e x t r a c t s Bu,Lhzm Bzophvs 4cta, IOs (197o) I32-I32

134

(. GUARNEROS, M V ORTEGA

If the e x p e r i m e n t was designed for the ~tudy of the kinetics of the reduction of the cysteine deaulfl~vdra~e activity, the inoculated media were i n c u b a t e d u n d e r appropriate condition~ and, at fixed tlme~, ~ample~ of them (lO-2O nil) were ~ l t h d r a w n Chloramphenicol u a~ i i n m e d l a t e l y added to a final c o n c e n t r a t i o n of 5o #g/ml, the ~amples were strongly shaken and placed in an ice bath The cell~ present in each sample were collected b \ centrlfugatlon in the c(~ld, washed once ~xlth o I M Trls buiter (pH 8 5) resuspended in the ~ame buffer, and the absorbance of the ~u~pen~lon at 66o n m wa~ d e t e r m i n e d in a B e c k m a n DB spectrophotometer These ah~orbance value~ were used for the d e t e r m I n a t m n of the u n i t . of abaorbance volunie for cal~ ulatum of the ~pet ~fic a c t i v i t y . f the enzyme preparation~

Tohtemzahon of th, cells To each ml of the bacterial ~uspenslon (with an ab~orbance between 2 and IO, read at 06o n m in the B e c k m a n DB spectrophotoineter), o oI rnl . f toluene ~ as added The sample wa~ transferred to an erlenmeyer flask of a volume at least IO trows greater t h a n t h a t of the sample, and the m i x t u r e was ~fiaken for 3o rain at 37" in ,~ r o t a t o r y shaker set at 24o r e v / n u n The toluemzed preparatlon~ were kept for no longer t h a n 8 h in an ~ce b a t h until they were u,ed Preparatzo~ of cell-free c ttracts The bacterial cells were induced u n d e r the different e x p e r i m e n t a l conditions described in the text and ~ ere harvested, washed a n d resu~pended in buffer a~ In the ca~e of tho~e used for toluenazation T h e y were disrupted b y sonicatIon for 3 lnln in a n MSE ultrasonic power u n i t set at I 2 A I n t a c t cells and debn~ were ~eparated b y c e n t r i f u g a t l o n of the ~omcate for 3 ° inln, at 4 °, at 3o ooo ~ g The clear s u p e r n a t a n t wa~ dialyzed in the cold (4 ~) for 12-16 h against 2oo tmles it~ volume of o I M T r > bufier (pH 8 5) The dialyzed extract~ ~ere kept at 4 ~ and used a~ such l)etermlnatzo~l of lhe cvstcznc desulJhs,dra~c and cvstathzonase actzvztle~ The cvsteine de~ulih} dra.e a n d cystathmona~e activities of the LT2 a n d SMIo3-2 ~traln~ were deternilned b y the colorlnietrlc m e a s u r e m e n t of the 2,4-dlnltrophenylhvdrazone of the e n z y m a t l e a l l y formed keto acid b v a ~llght modlficatum of the m e t h o d of \VIJE'~UNDERA AND ~VOOI)s 6 The i n c u b a t i o n n u x t u r e s contained, in a final vol ~f 2 nil IOO #mole~ of potassmni plm~phate buffer (pH 8 5), IO/~nioles of cysteine hydrochlorlde or 2o #moles of c y s t a t h m n i n e , IO/.naole~ of pyrldoxal phosphate, and either cell-free extract (o 3 to o 5 mg of protein) or toluene-treated cells (o 2 to o 5 nig, d r y weight) The samples were i n c u b a t e d 3 ° m i n at 37 °, and the enTymatw reaction was stopped b y the addition of o 5 ml of 25°0 (w/v) trichloroacetlc acid The control~ had the same composition, b u t the trichloroacetlc acid was added at zero time The precipitated protein or cell% were separated b y centrIfugatIon, and o I o 5-ml ahquot~ were t a k e n from the clear s u p e r n a t a n t These ~anlples were diluted to a final vol of 2 lnl with twice dmqtilled water, and then I ml of a o oi25_-a (x~:'v) ~olution of 2,4d I n i t r o p h e n y l h y d r a z I n e in 2 M HC1 wa~ added to each . a m p l e After a i o - m i n incub a t i o n at 37 '~, I nil of 2 5 M N a O H was added After d e v e l o p m e n t of the color for 15 n n n at rooln t e m p e r a t u r e , the absorbance of the sample~ was read at 45 ° nvI In a B e c k m a n DB spectrophotometer The cx.~telne desullhydrase activities of the S /yph,mufzum LT2 and E coh B BzoLhtm t3~ophi,. A~ta, 198 (1~)7o) 132 142

CYSTEINE DESULFHYDRASE OF S

t'vphtmt~rtum

135

a n d W strains were d e t e r m i n e d b y the m e a s u r e m e n t of either the k e t o acid or the H oS p r o d u c e d The i n t a c t or t o l u e n e - t r e a t e d cells or the cell-free e x t r a c t s were i n c u b a t e d in 13 m m x IOO m m tubes for 3 m m at 37 ° with ezther 1 2 o / m i l e s of Trls buffer (pH 8 5), for the S lh3'phnnurmm p r e p a r a t i o n s ) or I o o ¢tmoles of p o t a s s i u m p h o s p h a t e buffer (pH 7 2, for the E cob preparations) Then I 6 1 ~ i n o l e s ofcy~teine h v d r o c h l o r l d e were added, the t u b e s were h e r m e t i c a l l y sealed with parafilm-covered r u b b e r stoppers a n d the samples were I n c u b a t e d for 2 mAn at 37 ~ The final volume was I 7 nil W h e n the a c t i v i t y was m e a s u r e d b y the p r o d u c t i o n of k e t o acid, the reaction was s t o p p e d b v the a d d i t i o n of o 2 ml of a 25 ~}o (w/v) ~olution of trichloroacetAc acid, and the f o r m a t i o n a n d d e t e r m i n a t i o n of the corresponding p h e n v l h y d r a z o n e was carried out as before I f the e n z y m a t i c a c t i v i t y wa~ d e t e r m i n e d b y the m e a s u r e m e n t of the H.,S evolved, the reaction was s t o p p e d b y the a d d i t i o n of o I ml of a 12°. (w/v) solution of N a O H , a n d the p r o d u c t was t r a n s f o r m e d m t o m e t h y l e n e blue following the modification of S I E G E L 7 t o the m e t h o d of F o c o AND POPOWSKY 8 The specific activities are expressed aa functions of the nig of d r y weight, or mg of p r o t e i n (determined b y the m e t h o d of LAVXE ~) or units of a b s o r b a n c e volume (determined at 66o nni) present An the different assays Under the conditlon~ employed, I a b s o r b a n c e unit was equivalent to o 434 mg b a c t e r i a l d r y weight per ml RESULTs

Characterzzatzon of mutant SMIo3-2 In solid m e d i a m u t a n t SMIo3-2 grew only in the presence of cysteme, cystine, O-acetylserlne or l a n t h l o n i n e , the growth, h o ~ e v e r , was r a t h e r poor W h e n the m u t a n t was grown in m l m m a l glucose liquid m e d i A s u p p l e m e n t e d with either c y s t e m e or cystine, the g r o w t h s t o p p e d after some time unless new a m o u n t s of the corresponding aimno acid were a d d e d p e r i o d i c a l l y A t the same tAme a strong odor (,f h y d r o g e n sulfide was a p p a r e n t p r a c t i c a l l y from the b e g m n i n g of the e x p e r i m e n t I t a p p e a r e d as if the m u t a n t limited its g r o w t h b y d e s t r u c t i o n of the required a m m o acid Lanthionine, liou ever, allowed a continuous t h o u g h slow g r o w t h of the m u t a n t (FAg I) The fact t h a t the m u t a n t grew onl\ when either cysteine, cystAne or O-acet}lserAne were provided, while serine was inactive, i n d i c a t e d its lack of L-serine transacetyla~e a c t l w t y Tins was confirmed to be the ca~e when the activities of L-serine t r a n s a c e t y l a s e and O-acetyl-L-serine sulfllydrase were d e t e r m i n e d ~° m cell-flee extractq of b o t h p a r e n t a l a n d n a t a n t strains In the m u t a n t strain the first actAvlty was mAssing, while the second was s h g h t l y e n h a n c e d M u t a n t SMIo3-2 was, therefore, classified as a cysE- m u t a n t ~0

Some characterzstzcs of the cvstcnw desulflo,drase reaction of S (vphzmur, um pH optzmum F o r i n t a c t or t o l u e n e - t r e a t e d cells, as well as for cell-free extracts, the p H o p t i m u m for the desulfhydrase reaction was found to be a r o u n d 8 5 K~netzcs F o r a n y of the three systems m e n t i o n e d before, the d e a u l t h y d r a t i o n of the a m i n o acid was h n e a r for at least 6 mln The a c t i v i t y also showed h n e a r l t y with respect to e n z y m e c o n c e n t r a t i o n Slozchzometrv As s h o ~ n in Table I, a I I s t o l c h l o m e t r y in the p r o d u c t i o n of H~S and keto acid from the d e s u l f h y d r a t l o n of cysteine could n o t be d e m o n s t r a t e d , twice as m u c h HeS as keto acid x~as always d e t e r m i n e d No l a c t a t e d e h y d r o g e n a t e Bto~hzm t3zophys Acta, I98 (I97 o) 132-142

130

G G U A R N E R O S , 31 V O R I ' E ( , A

1 \BLE

[

STOICHIOMETRY

OF

FHE

CYbTELNE

DESULDHY1)RAbE

kCT[~ITY

OF

CELL-FREE

EXTR&CTS

OF

%

t3'phtmzt~ ~ m L'I 2 Cell~ w e r e g r o ~ n m q l u c o s e ( o 2 " , , , ~ / v ) h q m d l n e d m m , a e r o l n c a l l 3 a t ~7' a n d r e d u c e d u n d e r t h c ~ c o n d i t i o n s f o r 2 h in t h e p r e s e n c e o f 4 ° l t g / m i o f c . , s t m e

.qmoztnt oJ

P~oduct (lO~Z,)h ~)

/ht ol~ l tI

H2S

Ratio H=SI keto acid

]',CIO acid

(ITS) o

o o

11) 20 ~o 4 <) 5o

12 5 1q 2 2S 5 ~(~ ~ 47 .5

o o

t) () [~, 17 2~

3 ~ t b 7

I 2 2 2 2

98 02 17 24 ]8

07

/

¢E05 g tO

o

Na2S added (~lmoles)

O4

~>~100,

03

75 c

2 .

50

I /90°°o

o)

0

1

o

0

-

~

2

--)

3 4 5 6 7 8 9 10 )~ Incubation time (h)

o

20

40

Pyruvete

60

80

~00

added (~moles)

I~'l~ I G r o x ~ t h o f m u t a n t b M l o 3 - 2 m t h e p r e s e n c e o f c 3 s t l n e o r l a n t h l o n m e ( e l l s x~erc ~ r o , ~ n ox ~ 1m g h t a e r o b m a l l v , a t ~7', in g l u c o a e (o 4 o ~ / ~ )_( a s a m m o a c l d s (o 1%,, w/x ) - c y s t l n e (4 ° t~g/ ml) ( ells w e r e c o l l e c t c d a n d x~a s h e d x~l t h s t e r i l e s a h n e s o l u t i o n I n o c u l a t e d m e < h a ~ ere i n c u b a t e d a c r o b | c a l l v a t 37 ~ O , m l m m a l g l u c o s e , c , m i n i m a l g l u c o s e plus c v ~ t l n e (4 ¢)/~g/ml), • a n d ~7 m l m m a l g l u c o s e plz(s c y s t m e ( 4 o / , g / m l ) a t t h e t i m e s i n d i c a t e d b.~ t h e a r r o u s n e u a d d t t l o n s o f c ~ s t l n e (4 ° / t ~ / m l ) u e r e m a d e , m t h e h r s t c a s e o n l y o n e a d d i t i o n , ~ , m m m l a l g l u c o s e plus l a n t h l o m n e (27o l t g / m l ) l ' l g " I n t n b l t l o n o f c x s t e m c d c s u l l h y d r a s e a c t l x l t y o f c e l l - f r e e e x t r a c t s o f s lyph*mu*~um E l - ' l)\ i t s r e a c t i o n p r o d u c t s ( ' e l l - f r e e e x t r a c t s w e r e p r e p a r e d f r o m cell~ t h a t h a d b e e n r e d u c e d a e r o t n c a l l v f o r _" h a t ~7 m q l u c o s e (o 4 ° , . xx/v) c y s t m e ( 4 o l ~ g / m l ) h q m d m e d m m In the exp e r i m e n t s m x~hlch p y r u x a t e o r p v r u x a t e plus N H 4 ~ w e r e u s e d , t h e e n z y m a t m a c t l ~ l t x w a s follox~ed b y t h e p r o d u c t i o n o f H . b I n e x p e r i m e n t s m x~hlch N a a S w a s u ~ e d a s m h f l ~ t o r , t h e a c t l x ~ t y w a s m e a s u r e d h y t h e p r o d u c t i o n o f k e t o a m d P ; a c h s a m p l e c o n t a i n e d 25 t~g o f p r o t e i n Q , p \ ru~ a t e a l o n e , a t t h e i n d i c a t e d c o n c e n t r a t i o n s , ,, p y r u x a t e a t t h e m d m a t e d c o n c e n t r a t i o n % ph~s 1 o o / t m o l e s o f ( N H a ) . b O ~ , ~ , N a a > a t t h e m d m a t e d c o n c e n t r a t i o n s

]Jt~ehzm l~z,)/~hvs

qela l(),'~ (i~)7o) 132 t 4 2

C~ S T E I N E D F q U L F H Y D R A S E

OF S

typhtmurium

I37

actlvltv could be demonstrated in the cell-free extracts to account fl~r the lower amount of keto acid found lnhtb#zon b~' reactzon products Of the three products of the desulfllydratlon of cybteme (NHa, pyruvate and H2S), NH3, as NH4~, did not mhlblt the reaction up to a ioo mM concentration The effects of the reaction products upon the activity tn vtlro of the desulfhydrase are presented in Fig 2 The addition of increasing amounts of sodmm pyruvate produced mcrea~ed mhlbatlon, when the concentration of the inhibitor ua~ approx 6o times h~gher than that of the ~ubstrate (IOO vs I 6/,moles) there was a 40°0 inhibition The slmultaneou~ addition of NHa+ and pyruvate had, ho,~ ever, a ~trong inhibitory effect It should be pointed out that this strong inhibition was apparent only if the mhlbltors were added to the incubation mixture before the ~ubstrate, if they were added after the cystelne there was practically no lnhlbltlon S ~-, on the other hand, had a very marked mtnbltorv effect At a concentration equal to that of the substrate (I 6 #mole~), the inhibition wa~ practically complete If the inhibitor x~as eliminated by bubbhng nitrogen through the incubation mixture, practically all the activity ~as restored, lndmatlng that the lnlub~tmn bv S~ wa~ not due to enzyme denaturation Serlne and alanlne, at a 6o mM concentration, caused 8o°o and 5o"0 inhibition of the activity, respectavely, while threonme, mettuonlne and S-ethylcy~telne at the same concentration produced no inhibition

D~fferences between the desulflzydrase acttv~hes orS typhzmurzum and E colt When the cysteme desulfhydrase actlvltles of S t y p ] o , m t t r t u m LT2 and E colt B and W were compared, some differences were found (Table II) Intact cells of S tvphtmurtum desulfhydrated both cvsteme and cystme Toluene-treated cells lost the ablhty to attack cvstme while the desulfllydrataon of cysteme was enhanced In the case of E colt, all the desulfllydrase activity was l(,~t after the cell~ were treated with toluene Using intact cells, the addition of glucose to the mcubatmn media did not affect the desulfhydrase activity ofS tvphtmurtum (with either cystelne or cvstme q \BLE

I[

CYSTLINE ~ND E

DESULFHYDRASE

ACTIX I T Y I N I N I ~ C T e N D T O L E ' E N E - T R E A T E D

CELI S OF q

typJtyl}llt)llltkl

601l

Cells x~ere g r o w n a e r o b i c a l l y a t 37 ~ m g l u c o s e (o 4 ° 0 , w / v ) m i n i m a l m e d m m a n d r e d u c e d u n d e r t h e s e condxtlon~ for 2 h m the pre~ence of c w t m e (6o/~g/ml) W h e n m d m a t e d , i o o Hmole~ of g l u c o s e w e r e a d d e d t o t h e i n c u b a t i o n m i x t u r e s b p e c l f i c a c t l x l t y is e x p r e s s e d a s n r n o l e s of H 2 q produced per nnn per umt of absorbance volume

S lra ~n

S q E E E E

t'vpk~mu~zum L T 2 tvphtmurzum LT2 cob 13 coh 13 coh VV coh \ V

C'* lls

[ntact Toluemzed Intact loluemzed Intact Toluemzed

Speczfic actlvdv tn ttu p~esence of Cysh't m

Cvste tne plus glttc~s~

Cysh ne

Cysh ne plus chtcost

39 I42 9 o 2 o

45 146 27 o t2 o

13 I o o

15 2 o o

I i 3 o o o

Btochtm

2 i 5 o S o

--

Bzophvs Acta, I 0 8 (197 ° ) 132 142

I38

O GUARNEROh, M V

ORTE(,A

as aubstrates) while in the case of the E coh strains the a c t i v i t y was m a r k e d l y increased, as had been found by" ANDERSON AND JOHANSSON4 The p H o p t m m m for a c t i v i t y was also &flerent, as .~tated before for the S tvph> murtum activity, ~t was 8 5 while for t h a t of the E coh strains it was 7 2 A n o t h e r difference fimnd was t h a t in S t3'lSh,mur, um the cystelne desullhvdrase

and the cy~tatluonase actlvltles appear to be ditferent entitle> RO\VBURYANI)\~:O(ID~,11, working x~lth E coh, found t h a t the two afore-mentioned activities \~ere repressed a n d derepressed s i m u l t a n e o u q y From this observation they ~oncluded t h a t p r o b a b l y the same protein ,aas responsible for the two actavmes W h e n this po~s~ballty x~as studmd in the LT2 a n d SMxo3-2 strains of S @ph~murzum, it was found t h a t the t~ o activities b e h a v e d differently A , can be ,een from the d a t a presented m Table I I I , when tile cells were groxxn in the presence of cystme, tile ( v s t e m e desulthydrase a c t i v i t y increa,ed /5-fold while the c y s t a t h m n a , e act~v~t\ ~ as not affected, as con> pared ~x~th the actIvitie~ present in the LT2 strain grown m the ab,ence <,t"the reducer Al~o, when the cells were grow n an the presence of cystme and metln~mlne the level ,,f the cysteme desulthvdrase a c t i v i t y remained stable while t h a t of the ( v s t a t h u m a , e decreased m a r k e d l \

Inductwn and represszon o[ the cvstczne desulJhy,drasc achv~tv of S tvphtmurmm LT2 and E coh B Fig 3 qiow~ the kinetics of i n d u c t i o n of the cysteine desulflaydrase a c t i v i t y m ~ells of g lx'phzmur~um LT2 a n d E coh B growing m glucose I n both cases the a c t i w t y can be d e m o n s t r a t e d almost inimedaately after the addition of the inducer Ill

1 ~BI.E

CYSTEINE DESULlrHYDRASh. kNI) CYSIkTIIlONA.SE k C T I \ I T I E G OF CELL-FREE P.XTRkCT% OI" L ] 2 XND . G M I o ~ - 2 STRAINS (}F ,q l y p h t m u ~ z u m

Expt A (cllbweregrox~nacrobmall~ *or 1 2 h a t 37 m t h e m d m a t e d m e d h t (glucose (o4"o,x~/xx c . v s t m c (4o N
k~trmt

.f

C~ll.~ g~o~,n ~n

'~p~t~fit act~v~tv

slYal ]z

B

Cyst( zm ch ~ulfltvd* aac

C vstat,bz,mas~ *

1.1 2 LT2

(,lucosc Glucosc-c}stnm

o to ~ 57

I 2t t ~I

%M1o3-2

Glucosc-cxstmc

1 5 ()

1 22

Cvah zn~ d~ sltl/]lvd~ ase z*z th, presene~ ~g

Cv¢lal]l~oHa s~ *

%M~o3-2 %51io 3-2

* The

Glucose cxstlne (,lucose cystmemethlonme

(_ l'~lt llll

C ~'SI{ IHt pllt,~ tlt~tllll)~llH~

z 4o

-" 55

o .1o3

2 52

2 55

o o83

b p e c l h c a c t l x l t l e s a r c g l \ t.n c o n M d e r l n R t h a t

used in the re&orlon t¢~och¢m

t~toph3'~

4~ta, I9N (I97O) 13-' 142

the L-iSomer was

tht

(ll]lv substratc

CYSTEINE DESULFHYDRASE

typh~m~trhtm

139

\/o

80

g

OF S

30C

~o

_~ 20C o

4o

_o

10C 2= 90 03

50

100

150

pg bacter~cl dry wetght/ml

20C

0

50 100 Cystme concn ()Jg/ml)

150

F i g 3 Klnetic~ of m d u c t m n o f c y s t e m e desulfllydrase a c t l x l t y in 5 l},phzmumum L T 2 a n d t: coh B m glucose m i n i m a l m e d m m Cells x~ere grox~n a e r o b i c a l l y in glucose (o 2 % , x~/v) m i n i m a l

liquid m e d m m a n d r e d u c e d u n d e r t h e s e c o n d i t i o n s in t h e p r e s e n c e of c y s t l n e (4 ° / t g / m l ) E n z y m e u n i t s n m o l e s o f h y d r o g e n sulfide p r o d u c e d per n u n f- , k m e t m s o f m d u c t m n o f t h e q" l~,phzmur n l m actlx l t y , /~, k i n e t i c s o f i n d u c t i o n of t h e E coh a c t l x l t y F i g 4 E f f e c t o f i n d u c e r c o n c e n t r a t i o n in t h e i n d u c t i o n o f t h e c y s t e l n e d e s u l f h y d r a s e a c t w i t y o f 5 typhzmu~tzon L T 2 T h e e x p e r i m e n t a l c o n d i t i o n s ~ e r e as t h o s e s t a t e d in F i g 3, e x c e p t for t h e l n d m a t e d a m o u n t s of c v s t l n e used t h e t i m e o f i n d u c t i o n ~ a s 2 h Specific actl~ l t y is e x p r e s s e d a~ i n T a b l e I I I n s e t K m e t l c s o f i n d u c t i o n o f c y s t e l n e desulflavdrase actlx ltV o f 5 typhzmu~ntm L T 2 in glucose m e d i a ~ l t h either c ~ s t e l n e or c v s t m e ab Inducers E x p e r i m e n t ~ a ~ carried o u t a s t h e one reported in F i g 3, e x c e p t t h a t t h e c o n c e n t r a t i o n s o f i n d u c e r ~ e r e 66 l~g/ml for c ; s t e m e and 3 3 / ~ g / m l for c y s t l n e ~ ,, c ~ s t e m e as i n d u c e r , 0 , c y s t l n e as r e d u c e r

In S tvphzm.rzmn the activity increases steadily (for about 2 h) until the bacterial m a s s has approximately tripled and then dmunlsheb rather sharply A similar pattern was found when glycerol was used as carbon source although the acUvltv was a p p r o x m m t e l y one-third of that found in glucose W h e n the cells were induced under anaerobic conditions, using glucose as carbon source, similar results were obtained, but the activity of the desulthydrase was markedly lower, approximately one-fifth of that obtained in aeroblosis In the case of E coh the increase in the cystelne desulfhydrase activity during reduction m glucose-minimal m e d i u m was faster than In the case of S (vphzm.rutm, the m a x i m u m act~wty u as reached before the initial bacterial mass increased by onehalf (betu een 3o and 45 m m after the addition of the reducer), the activity declined gradually as if it were diluted among the increasing bacterial population W h e n the induction was made in glycerol minimal m e d i u m , the kinetics of induction of the activity followed v e r y closely that obtained with glucose W h e n the mductlon of the activity in S t vphzmurn~m .aas determined after a fixed time of induction (2 h) with variable concentrations of Inducer, it was found that the de~ulfhydrase activity increased hnearly with the concentratmn of the inducer until it reached a plateau A further increase in the Inducer concentration did not increase the actlvitv (Fig 4) The same results were obtained either with glucose o~ glycerol as carbon sources W h e n short periods of induction were used, it wab found that the kinetics of reduction were the same either with cysteine or cystlne (Inset, Fig 4) Bzochzm Bzophvs :hta, I9V; (197 o) 1.32 142

I4o

G

1 kBLR

GUA-RNERO%, M

V

ORTEGA

1\

REPRLbSION BY T R Y P F O X E OI, T H ~ f. "t %TEINE I ) B b U L b H Y D R ~ b E a.CTIX ITY Ob % [U/}hlD1Hl lHDI L ] 2 ( e l l ' , ; ~ e r e gr{B~n m glvccr{}l (o 2 " o , x~/\} n l l n l n l a l h q m { 1 m e { h u m d l l d i n d u c e d Ill t h e r e ( h e a t e d nRdld (~lucose and glycerol, 0 2%, x\/\ tryptonc, I {}o, W / \ c T h t l n e , 8 0 g~{/ml~, a e r o l ) t c a l l v a t 37 \ t m t c r x , t l s , t h q u o t s \~crc \ \ a t h d r a x ~ n a n d t o l u e n e - t r e a t e d celG \\ere tncp,tred ~,p{ctflc , t c t B ~ t w s a r c e x p r e s s e d a s m I a b l c I1 ] lldllctlo]l Dlt dl ttDI

{,lucobe q %stme (.Iucosc t tr 5ptonc (ducobe }- t r y p t o n e Glycerol 4 c~,~tme Glycerol ; trTptonc (,lvcerol ~ tryptonc lr~ptonc ~ c)stmc

d

cxstlm

} cx, s t m e

lh

2h

237 12

4Ii

172

I3I

276 9 ~0~ ~5,";

-~2z

\ \ Ith tryptone as carbon and energy s o m c e , the deaulflwdrase activity of 5

(vphzmurmm was repressed, even when the concentration of the inducer was increased This repression was not affected by the addition of either glucose or glycerol (Table IV) The results (not shown) obtained m studying the reduction of the cyateme debulflavdrase activity in tryptone m e d i u m fl)r E cos B were smular to those obtained by ANDERSON AND JOHANSsON 4 f o r the E coh ,train Crookea the actavltv was greater in the absence than m the presence of glucose Finally, N H 4 ~, pyruvate and glucose had a stxmulator 3 effect on the lnducnon of the cysteine desullhydrase a c t s ltv of S (vplmnurzum up to concentratlon~ of 3 o, 2o and 4 ° raM, respectively (Table V) 1 ABI.F

\

lsl, bFCT O1, \ A R I ~ B L E CO.NCFNTRXIION% OF GLUCOSE P Y R U X A r E ANY) N H 4 4 ()1, C'~B'IlrlNE lkEGULFHX~I)RASE A C I I X I T Y O1, % &V]b]IIDIIIllIIIll L ' I 2

ON TIlE I N D U C T I O N

kxpt A ( cll~ ~ e r e g r m ~ n i n g l u c o s e (o 2 ° , , , x~/~) h q m d m e d m m , aerolncal] 5 at ]7 , induction V, a s c a r r i e d o l l t f o r 2 h i n IDlnllTldd l l l e d l u n l ",~lth lOO [1~4/II11 o f o , ~ t l n e a n d t h e lndlcdt{_d c o n c e n tratmns of glucost Expt B ( e l l s ~ e r e g r o ~ n i n p v r u x a t e (o 22 % , x~ ] \ ) m i n i m a l l l q m d m e c h u m , , t e r o l n c d l l \ a t 37 , l l l d u c t l { } n ~&~ c a r r i e d o u t f o r 2 h ill illlnllll&l l n e d l l l l l l \ \ l t t l i o o iig,/inl o i C \ S t l l / e ,rod the lndmated ,unounts of pbruTate Expt C ( e l l s \ ~ c r c g r o \ ~ n ,ts m E x p t ~_, r e d u c t i o n \ ~ a s calrmd out for 2 h m mmnnal medmm \~lthout Nft,~ \ x q t h t{lucose (o 2{}~}, ~,lx} a n d c~. s t m e (~oo Ilia/roll, a n d t h e m ( h c a t e d a m o u n t s o f ( N H a ) a q ( ) ~ I n all c a ~ e s t h e p r e p a r a t i o n o f t o l u e n e t r e a t e d c e l l s all{1 t h e d e t e r m i n a t i o n o f e n z y n l e a c t s l t m s xx ~ r e c a r r i e d o u t a s i n d i c a t e d i n MATFRI KL% ~,.NI} M E T I I O D , ¢~pcclfiC dctlx, l t l e s tilt ~lXLI] &S 111 t h e e x p e r i m e n t of Tal}le II

12tpt A

L~pt B

(dmose ( , t ll )

bptczfic acl~t, ltT,

I .5 ~O 20 4° BO ~oo

4{i i 21I 7 2I 90 2~5 4 293 ~ 2907 242 t) I97 8

l~o~htm

%dzum pl,~ m'ah ( m ll )

t 5 IO 2O 4° 00 IOO

l.t/~t

C

%]*tttflc aclletlv

(i\'H4) z~'04 ( ~ 3¢[)

Speczfic atht,tly

2~ 74 124 l~q ~39 TI 5 03 II 5

7 5 l o ", 7 5 TO--1 7 5 15 75 I50

1Q2 198 198 2OI 205 ]O5 I28

~ ° 5 2 () 8 I

B~ophvs ALia, I08 ( I 9 7 o) 132 i 4 2

(i 4 1 6 7 3 ,~

CYSTEINE DESULFHYDRASE OF S typhzmurtmn

14I

DI~CUbbION The results p r e s e n t e d in this p a p e r establish some s l n n l a n t i e s a n d differences between the cybtelne desulfllydrase activities of S tvph~muru¢m a n d E colt I n both organisms the e n z v m a t l c a c t i v i t y is not repressed by" glucose a n d for the one of S lyphzmurzmu there is also no repression b y either p y r u v a t e or NH4+ , end p r o d u c t s of its activity" W h i l e the i n d u c t i o n of the a c t l w t y In b o t h organisms follows m l t m l l y the same p a t t e r n , there is a clear difference in the permanence of the e n z y m e after the m a x i m a l a c t i v i t y has been reached In the case of the S typhzmttr, um activity, there is a s h a r p decrease of It, p r o b a b l y due to r a p i d i n a c t i v a t i o n of the enzynle In the condltlons used F o r t h a t of E colt it a p p e a r s as if the enzyme stops being synthesized and then 1~ d i l u t e d along m the Increasing p o p u l a t i o n There 1~ also a difference in the induction and repression of the cystelne desnlfhydrase a n d cy~tathlonase activities \Vhlle it has been r e p o r t e d u t h a t In E colt both activities were repressed and derepressed simultaneously, m the S tvphwaurtum >tralns used m the present w o r k both activities were induced and repressed independently There I~ also a difference in the s t a b l h t y of the e n z y m e tox~ards toluene I n the case of E coh the d e s u l i h y d r a t I n g a c t i v i t y is t o t a l l y lost after toluenlzation of the cella vxhlle in S @phtmurzum the a c t i v i t y t o w a r d s cy~telne is enhanced after the t r e a t n i e n t However, the toluenized cells of the l a t t e r organisms lost the a b i l i t y to d e s u l t h y d r a t e cystlne, indicating t h a t the cvstme reductabe necessary for the f o r m a t i o n of cystelne from c y s t m e is lost by" this t r e a t m e n t I n i n t a c t cells, the presence of glucose lncrease~ the desulfllydration of cystelne b y E colt cells while it has no effect in the activity" of the S lvphzmurtum cells, this could indicate t h a t the m e c h a n i s m of e n t r a n c e of the a m i n o acid Into the cells IS different m the two organl>nis As expected, the cystelne desulfllydrase a c t i v i t y of S l y p t u n m r m m was mark e d l y i n h i b i t e d m vtlro b y S 2- or by" a n u x t u r e of p y r u v a t e and NH4 + During its activity', hox~ever, no I I s t o l c h l o m e t r y in the p r o d u c t i o n of H2S a n d keto acid could be d e m o n s t r a t e d , i n s t e a d an excess of the first c o m p o u n d wa~ always found A l t h o u g h no s a t i s f a c t o r y e x p l a n a t i o n for this a n o m a l y can be given {no d i s a p p e a r a n c e of p y r u v a t e due to r e m n a n t lactic d e h y d r o g e n a s e a c t i v i t y could be d e m o n s t r a t e d ) , the p o s s i b l l l t y r e m a i n s t h a t a n o n - e n z y m a t i c reaction between the keto acid formed and the cystelne present could have t a k e n place 12, a l t h o u g h no test was done to prove it This s i t u a t i o n is similar to t h a t found b y ANDERSON AND JOHANSSON4 with the cystelne desulfhydrase of E cola m which, depending on the e x p e r i m e n t a l conditions used, there was a g r e a t e r p r o d u c t i o n of H2S {with r e g a r d to NH4+ ) t h a n the I I stolchiolnetry e x p e c t e d from the reaction ACKNOWLEDGMENTb W e are i n d e b t e d to E R Squibb a n d Sons de M6xico for generous gifts of antlblotIc~ We t h a n k Mr Jose Chavez fl)r his technical assistance REFERENCES I J O LaMPEN, R R ROEPKE AND l~I J JONES,3rch Bzochem , 13 (I947) 55 P H ABELSON, D B CO~IE, E T BOLTON AND R J BRITTEN, Carnegze Inst Wash Pz,bl , 607 (1955) 390

2 R B ROBERTS,

Bwchzm Bwphvs Acta, 198 (197° ) 132 142

142 3 4 5 o 7 8 o zo II 12 I~

G G U A R N E R O b , M V ORTEGA

,P DESNUIsLLE ~ND (- FROMAGEOT, E*~ymologza, O (I939) So D (_ ANDERSON AND I~ ])t JOHANSSON, J (;~n ~Izcrobzol, 3o (I9(~,) 4S5 ]~I D DA'vlS M',D E b MINGIOLI, [ Bactemol, 6o (195o) i 7 ~ X.VIJESUNDERA ~ND D D WOODS, J Oen 3hcr,~b*ol, 20 (19o2) ~,53 L M SIEGEL, :lnal Btochem, I I (1965) 126 J b2 FOGO AND M POPOV~SKY, Anal Chem , 21 (1949) 732 g LAYNE, in S P COLOWICK AND N O KAPLA.N, ~,lIethods z~ Lnz3'mology, Vol 3, h c a d e n I K P r e s s , Ne~; Y o r k , 1957, p 447 N 3,I KREDICH AND G M TOMKINS, J B~ol Che~*i , 24t (1906) 4955 R I~O'~BURY AND D D WOODS, ] Gen ~lI~c~obzol , 35 (1904) 145 A DUGAICZYK, M T M~LECKI AND J J EILER, J Bz,,l Chum , 24:; (19081 22~;0 R g ROBERTS, P H ABELSON, D B COVvlE, E T BO£TON kND f{ J BRIFTEN, ( a ~ z , g ~ lnst IVa~h P u b l , 6o 7 (1955) 38~

Bzochlm Btophys .4cta, 198 (197 ° ) I 3 2 - 1 4 2