\ 'ollll"c ' : ~--;, i\' 1I IIIIwi 1 /'1/11/, ',1 01 11, \ . 1.

Inhibition by Sulfonamides of the Biosynthesis

of Folic Acid I

Gene M. Brown ~

'. Vood ~ (~~) laid till' g nllllHl w ork lor ti ll' [,llwida t io I'1 of a hiolog ic flln ct ioll fo r p . <l minol )(, lI zo ic ac id ( p-AB ) w ith ti ll' oh~(' r ­

\'a ti o n thal th is CO lllP01111d a lltag(lili /.(·, th e hadcri os la ti l' prope rt ies o f Sl tI fOli a m id e~, Laler work cs tahli shed lha t p-,\ B is a s l ruc­tu ra l unit o f a m o re complex co mpo llnd , foli c ac id (I . \I ) . N o evidencc cxis ls to indicate lha t p-A B is 11C('(1cd for a ny pm­pose o lhe r th a ll as a prccm so r o f fo li c ac id . \Vork w ith int act b acter ia l ce ll s h ad ('s la h ­li shed tha t s ltlfon <l mieles in ll'r/('I"(' \\ ith the cellular sy nth es is o f fol ic aci el ( 10, 1~ . ~::),

and tha t p-A13 r cv(']"ses th is illhihilio n ill a compe titi ve m a nncr. T he e lu c id a ti o n of the c n z"m a ti c rcactions th a t res ult in th e bio­synth es is of foli c a c id b " Shiota a mI cow o rk ers ( I:; I ~) and 11}' B row n a nd hi s coll a110rat'o rs (:i . (;, 2~) provided an op­portunit y to Stllel y the crkc ts o f slilfon <l­miel (,s Oil th e c nzym a ti c utili zalion o f p-A B for the fo rm a tio n of foli c ac id a ncl re lat ed com p 0 1m cl s.

This p a pc r \\ 'ill h e presC'nt ed in ti ll' for m of a shorl j'( ' \ 'iew in w hich the hi os\'n thesis o f foli c ac id will h e di sc li ssed l)ri (: tk 1'01-lowcd h y a som ew ha t m ore deta i I(:d d e­sc r iption o f the work w ith sillfona m id es .

Enzymatic synthesis of folic acid and related compounds. f oli c aci cl cO llt a iliS three s tru cl ilra l unit s: a pter idinc mf)idy, p ­AB, and g lut a mi c: ac id, \fos t o f the ~ i g­

nih canl work th a t has led to a n lInd e r­s ta nding o f th e en Z ~ ' l1le sys te m th a l ca Ll­lyzes th e s~ ' nth esis o f th e vit a min from these precurso rs h as com e from th l' bl ll)­ra to ri es o f Shio la ( I ~''') a nd o f 111'0\\ ' 11

1 P rcsell led at S)mpmilli ll 011 S, tl fll l' l'~, ( )'i" j ;,, ""1 Coo pera l i\c :' ferii,:tI Sril' l1 ((' P rogl ;lll l , 'i: 1I 1 "' ;111· , i,co. C al ifo rni :l , II ]\ ray 1%7.

"G . . \1 . II ro\\'n , . Pit . D., Divisioll III' II ill, II (' m is( 1 y, DCJ)~rl lll e ll t of BIOlogy, MassaLi lllsclI' lml ilu le of Technology, C:l lll iJlirlge, ;.. rassachusells 02 139.

580

(.-, . • ;. ~ " ) . TIll'w \\ o rkns II sl,(L rcs pective­k , ce ll - frl'(' ('x trac(s o f l ,flc/O/}( II· illll .\' /I7all ­/ fl /,/IIII a lit! ('x trad, of I ~scl l(' r i cllifl ('o f i as .' Ollrces or (' Il /.YIl H'S . I t has hCt' 1i es ta hli s lwcl tll a t. in bot h ,, 's te lil s ( ';' \I ;). th e pt c ridillt, IIScd as Sllhs tra te is 2-a miIl0--l -h n lroxv-6-hydroxY IlH' th y l-i , i-l- clih ydroptlT idill c ( ji ig . 1 ) . This compo llilel ca ll 1)(' cOIl\ 'C' rl ed en ­zY lil a li ca ll ~ ' to e itll (' " dih n lropteroi c ac id ( ill the prC'i( ' Il C(' of p-.'\B as C'osul)strate ) o r diil ydrofoli (' ac id ( ill ti ll' pn 'se ll c(' o r p­a lilin obc ll /.O\ ·lg luta lni c ac id ) o n1\ ' \\ ·he ll :\ TI' a lld m;lgn('s iulll iOll s a re a ls(; present ill th e reac ti o ll mi xtmes. \\'c is ill a n a nd 1\1'0\\'11 ( ~" ) ha\ 'c fr :iC'l ioll a tcd ex trac ts of E. ('o li a nd obt a incd t\\ ·o en zy me frac ti on s ( fracti o ns !\ a lld 13 ), h o th of \\'h ich a rc nced ed fo r the form a tioll of dih yd roptc ro ic ac id from p -I\ B, I\TP a nd the dih yclrople ri ­dill c suhstrat e. 011 1' of these c n/.)' llll' fra c­tioll s ( fra ctio n i\ ) is lI eeeled to ca ta lYz(' til<' form a tion fro lll ti l(' dih yciropt e ridill e a lld 1\'1'1' of a s lIhs la ll C(' th a t ('; 111 h e ('oll ve rt ed by the secolld c n/X Il1l' frat'l ion ( fract iOIl B ) to e!ih n lropt e roa te ill th e pres( 'ncc of p-.-\B . Shi o ta eI al. (l ~ ' I' ) haH' svnth cs izcci I he m onophos pho a nd pno ph os j)h o es te rs of 2-a m i no--+-h yd roxy-6-h yd roxY lll e t h \ 'l-d i­h ydropt e rid ine a nel showcd th a t in thc L­,llall/antm sys te m the pyroph os phat e estn, hilt Ilot th e mOllophos pll a te , can h e eOIl ­\ ('['teel to dih ydrople roa tl' in the prese l1 ce or p-AB alld ill th e a h se ll ce o f !\TP. \Ve ism:lll aile! BrO\n l ( ~" ) th e ll fOlilld tha t th e ir frac ti o n 13 , p rc p a red from 7':. co li ex tracts, could ca ta k ze th l' snltill's is o f clih n lroplC'r­oak from p -.'\ B a lld thc p)To pllOsph a te es te r, hill th e 1l1 01l0pllOsph a tc es te r was Il o t IItili zed as s llhst ra te 11\' e itit e r fra ct ioll B a IOlH' , or ill th e pn's(' l;('(' of ATI' a llt! hoth fractiolls 1\ a lld B. Th('se r(,Sltlt s sll g ­gcs tcd lh a l tit t' p\T(J ph os phatc es ll'r is all illt cr llw dia t(' ill til e p a lh \\'ay a lld th a l it s

" ,.., I (11 t ,) ) . I.") , - . --' HrulC II : III/, ill i/ioll h !f SII I/IJII(flllirics 581

('H

I -0 C

' I Y

I

I H

2 - Am ,no- 4 - hyd , o xy - 6-hyd ro xy -

o /I

o II

me l hy l-7, 8 - d lhyd rop1e r idlne / '

Py rophosphale .

OH I

p- Am inobenzoyl -

glu l a m lC aCid

/'C N N /' 'C/ ""' C -C H

I II I " - NH --< )-COO H

/C-'::::- /C"" /C HZ HZN N N

Py rop hos phale

Glu l amlC aCI d

:;

1

ATP Mq K 1 ,

I H

Dihy drap lerOIC acid

I.

Dlh ydr o fo l lC aCid

FI C , J, The e nzym a l ie ]'( 'ad ions I ha l lead 10 I he hios\'nth es is or tlih ytlro /'o li c ac id ,

formatioll and sllhseqllell t IIt ili l',atioll for til<.' ('Il/ymatic s\'llthcs is of dih~'dropteroa[(' is as sh()\\'I1 ill Fi g m c 1,

,'\I so SIIO\\,1 1 ill Fig llre l is til(' rca clioll \\'hcreh\ ' diitnlroptc roi c ac id a lld g lu tamic acid a re utili zcd for the fOnl1a tion of di ­hydrofolic ac id , The (' nl'Y me that ca tal yzes this reaction h as becn found in ext racts of a large numhe r of mic roorga nisms ( ~ ), in ­c ludin g slI ch cli n'rs(' \'ari (' ti es as yeas t, .\'( ' /II'OS/lO/'{/ , ,\l!I('O/JII('/('I'illlll IIrilllll , and \ 'arious spcc ies of cnll'rohactn ia, The e n­z\ 'n1<' h as I)('cll pmified som(' ;3.,)-fold from ex tract's of E, coli a nd some of it s propcrti(,s d escrihed ( ~ ) , As ShO\\'ll in Fi g lll'c I , AT P, \[ g ' a lld Ill ol lo \'alcnt ca tion ( ci tit n "- ! or N II , I ) a re a II need ed for tit is c l1 l',ynw to he ac ti\ 'l' , Ne ithe r pt e roi c acid nor tl'trah ydropt c roi c ac id call Iw Ii sed ill pla C(' of clilwdropt (' roi c ac id as s ll!>stra te,

i\ I t I ;ollg h p-a III i noh c nzo\' Ig llI t a 111 it' ac i d p-,\I1C ) can 1)(' IItili l',cd ill plaCI ' of p-;\B for th e direc t form;ltion o f dill ydro fo li c:

acid ( I; 1 ~.), it is fe ll th a l p-:\ Be: is proh­a!>'" not of importan cc :1 :~ an int e rll1ediate in thc hios\' lItlH's is of foli C' acid compollnds , s ilJ('(' p -:\BC is no l utili l',ed as dkd i ve l~ ' as p-:\ B ill most s \ 's tems and 11 0 c \'idc nce could 1)(' ohtailled thal p-A Be: is a proclud form cd cn zY l11ati call~' from p-:\13 and g lu ­tamat e in E, ('oli ( ti ) ,

Th e g( 'lI e ra l importancc of th e path\\'a\' g iH'n in Fi g ure I is c \ 'ide nt in the fa c t that til(' reactions for the synthes is of dih ydro­[lt e roi c a c id a rt' known to occur in a widc \ 'ari (' t~' of organ isms, inc ludin g ( in addi ­tion to L /l11l1l/lInl/ll alld ,,; , co li ) pneumo­cocci ( 1 ",~ l ), mi crococci ( ~ ), yeast (8 ), Pekill c ll l is hf(lss ica ( II ). X CII/'Os/l om Cl), '\{/Ico/}(f c /eri lllll II v i/llll. (:1), Bllc illll s SlI1J/ilis

(:1). COI'I/Ilc/}(Ic /e rili/H s/l , (:1), e ll/ e /'()h(/(' ­

/cr ill (:1) s \l(·h as Pm/Cll s I/lOrg alli , !\ c ro ­

/ )(II' ! I'/' W' /'{J i!J' I/('S, and SlIllIIonel/a /!fIJl/i ­

IIII/rill/II , alld '" i('mco(' (' /I S lysor/e ik/ic lI s (:'),

Inhibition by sulfonamidcs. The effects of ~ lIlro ll al11 id t ·s on the l' lI l',YlIlali l' synt'lwsis of

582 lllf ('/'Il(lfioll(ll ] 0 /1/'11111 of L(' I )J'{) ·\· ~I J967

CJl

::l 0 1.2

~. w u => 0 1.0 0 0:: SULFANILA MIDE a... (J) 0.8 f-z W ---1 :; => 8 0.4

w ~ 0 .2 ---1 o LL

SULFADIAZINE

20 40 60 80 100 120

HALF MA XIMAL

INHIBITION

.~

140 160 180

•

SULFA DRUG, mJ-L MOLES PER ML.

Flc. 2. The elk-cis of sulra dru gs Oil the cJJI:yma ti c S\' lIth l' .-; is or f()li c ac id. Tilt' CO II ­

te llt s of the rcactic)I] llli xtllres a re g iw'lI ill the text. Details of this l'x p erinH'lIt ca n be found in a pa pe r hI ' BrowlI ( I ).

dihydropteroic ac id (expressed as "fo late equiva lents" produced ) hy an enzyme sys­tem from E. coli a rt' shOln1 in Fig lll'e 2. Each reaction mixture contained : 2-amino - -4- - hydroxy - 6 - h ydroxymethylc1ih~ ' -c1ropte ridine, A TP, \ I g - '. , the enzyme prep ­a ration , p-A B (.5 mfLmoles per mI. ), and sulfa drug as shown in the hgure. 111e three sulfa drugs tes ted were all e ffective as inJ1ibitors of product formation, hut the three were not equall y e ffecti ve. Su lfadiazine was mos t ef­fective and sulfanilam ide the leas t. Sulfa nili c acid is, of course, not a sulfona mide , lmt nevertheless is a potent inhihitor. The dotteel line shown in Figure 2 represents half-maxi­mal synthesis of product under th e ex peri­menta I conditions. A useful term that has heen used to characte lize the e ffects of compe titive inhibitors in biologic systems is the "inhibition index." This refers to the ra ­tio of the concentration of the inhihitor to the concentration of the substrall' ( with which it competes) that results in half-maxi­mal synthes is of product where maximal syn­thesis of product is D1eas1U'ed under the

same experimental con ditions in the ab­sence of inhillitor. For exam ple, from t he data of F ig llrc 2 it is poss ibl e to ca lcula te inhibition indices of approxi mately 2.0, 3.0 and 20 for sulfadiazine. sulfanilic acid and sui fan i Lt m ide, res pect i \.e1y.

In an experimcnt where the efkct of a compe titive inhibitor on bacter ia l growth i~ mcasured, the inhibition index is clt'iln ecl ill the sa me way except th at growth is the process measllred qu ant itat ive ly rathl'!' than the formation of a produ ct. A nllmher of sulfonamides have been analvzed for th eir ahilities to com pete with p~.-\B alld thll s to inhibit the gro\vth of E . ('o li , and al so for the ir propert ies as inhihitors of the IItiliza tion of p -AB for the enzymatic pro­duction of dihydropteroi c alld b y an ell ­zyme syst('m prepared from E. coli ( I ) . !\ li sting of tIl(' ('a bllateel inhihitioll indi c('s determincd l)()tll ill g rowth and enzymati c expe riment is g iven ill Table 1. For tIl(' deta il s of this ex per inl l' llt , the rt 'adcr is referred to a paper b y Brown ( I ). I t is evident from the data of Tahle 1 that a partielli ar sulfonamide is a mu ch better

BrOlVlI : ] II/,i ll ilioll li!/ Sll l/OII(lIl1ides 38,3

'1',\ 1l 1.1'; I , /,'.!Ft-d il '('III'-'S of -, "/fIJI/(/lI/ itil''' us illhilil'/o rl;; of Ilt l' (//'O/I 'l iI (If i ~ , co li and 0" inh i ll /t()rs oI till' ( ' 11 ::1/1110/ 1('1"(//10/ i ll II I~r II)/III( ',

Illh illil()i'

;-;11 I fa thiaz. ".' ;-;ldhhnll z:u ll i. I., ;-;lIifanll'l':I zill (' :-ili lfaJ iazine :-ililfanilic acid ;-;lIlfall1 (' t ha7. ill (' ;-;ulfapy ridill e ;-;lIifagufL llidin. ' ;-;lIlffLcc lalll iil( ' ;-;1I1 fan ila III i( 1(' :-i lll fa';lI xiti ill l' Jl - :lIll iIl O~a li ('d i (' :If 'id

I-:II Z,\ ' -

1ll :l l i.,

(I , I () () ~ j( I

I 'J

, ~ I

~ " ~ ~ I :u.; ;) ,U

1 ,0 ~ll.l)

~;l II ~-I ()

( :ru\\l ll

~;-)()

I , !(I()

~I II

:no

(" r J_" l , -1()()

1 G , ;)()() :? , !JOO

21 , ,')00 Ii , ,',Ot)

(i-' , (lOll

inhibito r at the e ll zyme 1(; \ ,(; 1 than it is as an inhibitor of bactc rial growth ,

A comparison of th(' inhibito ry properti('s of the sulfonamides indi cates that, in g(, l1 -eral, jf a sl1lfonamid e is a good grow th inhibito r it is a lso a relativel y good inhibitor of the C'1l zyma ti c read iOIl , a I t hOl1 gh some exceptio ll s are ('v ide llt ( 1'/' sl1l fahell -zamide, sl1lfag l1 a llidin(' , s l1lFasl1xidillt' ), Two compounds that were tes ted , sl1lFanili c ac id a nd p -amino-sa li cy li c acid , arc not suifonamie!es but were ill c luded because 01, the ir simi la riti es in s trllc:tme 1'<) ]1 -A13, Sul ­fanilic acid was co mpl etely ineffecti ve in inhibiting bacteri a l g rowth , but was quite effective as a ll illhihi tor in th e e ll zyma ti c experiments , This s uggests tha t thc cell s a re not permeahle: to thl' hi g hl y negative ly chargcd sulfanili c acid , T he fact that a ll of the sulfonamides werc far h (' tt er inhihitors of t he enzymatic synthesis 01 dih ydrop­teroa te than they w('re of th e growth of 1~, coli sugges ts that these hacte ri a are far less permc<l ble to thew compounds .th:l1l they are to p -Afl .

From the data pres(' nt n l ill Table I , itis evident that sulfa thiazole is the most c lrce­tive of a ll the slIlfonamides as an a ntagonist of the utilization of p -AB , In o rde r to determine w hether the observed inhib ition

of thc fOJ'Jna tio li o r p rodlJd \\as CO JJlpdi ­ti\' (' , a Illlm lll' r of r('act ion m ixtllres \\T re prep an'c1 to ('o lltaill \ ';II-y in g eOll l'(' lItr a tio Jl o.; of hoth p -:\B ;lIld sili fa thi azn !c, .In OJ\( ' s(Til's, ;1 constan t a ln Olll lt of s lIifat hiazok \\'as add ed to eac h llIi xtlJl'l ' alld the conet' lI ­tratioll of p -:\ B \\' as \'a ri l'd , Thr('e ot l)(' r s ll ch se ri('s o f r('act iOIl III ixt II res \\ '1' 1'('

prepa red , ('aeh se ri cs ('o lltaini ll <' a dUfl' I'( 'lIt alllOll l'Jl of slIlfathi ;lzo )e , Th~ d a ta ; 11'( '

PI'( 'sl' lil (' d ill Fig llJ'( ' :3 as dOIl],I( , r('cipnw;t! p lots ( ti ll' sll-l'; tikd LiIH'\\'l'a\ '(' r-Burk c plot ) th at a r(' traditiollall ), II sl'd to d('tl'l' ­mill( ~ \\'hl'lher tl w inhihition is compL'li li\'l " The J'( 's ,titing fami ly 01 straight lim's, each lill e w ith a di/fl' l ... nt SIOPl' hu t th l' saill c inLl'rcept , indicall's th at th l' illhihit iOIl h \ sulfa thiazo le \\ 'as compdi ti\ l' \\ 'it h p-AI\' '

JIl o rde r to confirm that compditi\'l' inhi ­hition killeti cs appli l' d to till' S),S tl'Jl) dl' ­scri hed a ho \'c, exper i Illl' lI ts \\T re d esig ned III \\'hi ch r('actioll mixtllJ'('s \\T IT pn 'par('d to contain slllfa thi ;l'l.oie ami CVl'l'Y com­pOlle nt ( in c illdillg th (' l' II /,y me sys'tc m ) n('ed ed Jor the ('n zyma t ic produc tion of dih )'droptcroat e , L'xcept lor p -.-\l3, After a pre liminary inc llha tioJl pe riod ( :37 C ) or t\\ 'o ho lJJ's, p -AB was thl' ll a dded to the readioll Illi xt llre ane! ill cllbat ion \\'as CO il ­tinu cd fo r a ll o th (' r 1\\'0 ho m s, Control cx­perime nt s illdicat ed that the a mollnt 01 p-AB add('d \\ 'as g n'at eno ll g h so that il it had becn added at the sa mc tim c as th e sllifathia zo le, no inhihitioll by the drug \\ 'llltid havc heen e \ 'ide nl. .It was expected that if strictl y CO lllpl'liti vl' inhibition kine t­ics wcrc foII O\\'l'd , the prelimina ry in clI ­bat ion in the prcsence of the s ulfathiazo le a nd the ahs('nce of p -AJ3 wou ld have no elfec t on thl' production of dihydropteJ'oa te during th l' s('cone! in cuha tion pe riod ( a fte r the a ddition of the p-A13 ) . J] 0\\'(,\'l'J', the n 's lJits sho\\ 'n ill Table 2 indicate th at the pre lim inary incuba tion with sulfathiazo le rcsulted in inhibition that \\ 'as !lot rcve rsed hy subseqll cnt addition of p -AB ( Heaction ,\Iixturc .,I , Table 2 ) , Heac tion ~ Ii x ture 2 of Table> 2 shows tha t when both p -A13 and sulfathiazole \\'l'J'C added t ogether , th(' amO Ill)t of p-l\B add ed ",a s sllllleicnt so tha t' w ry littl e illhihition occllITed ( eoJl1 -pare w ith Heact ion ~ I ixture 1 ) , Thc ('O'('c t of the leng th of the firs t ineubation p e riod

,51H Ilit erlliltio llill j U/l1'1I111 (If I.C)/'()S!! ] ~)67

z 0 f-U <! O.S -W 0:: LL 0 04 >-f-

U 0 --.-l 0 ,3 W O,OOSM 5T > LL 0

~ <! u 0 0:: n... 0 . 1 -u W 0::

20 4 0 60 80 100 120 140 I

[s] Fl c. ,'3. A double recip rocl l p lot of J Isubstrate aga inst l i veloci ty of th e react ion in

the prese nce of va rious concen tra tions of sulfa thi azole . [S ] = The conce ntra ting of p­A il ; ST = sulfath iazo le. Thc other components of th e reaction m ixtures were: 2-amino-4 -h ydrox~'-6-h ydroxyme th vldih ydropteri din e, ATP, i\ r gCI ~ , and enzyme prepara­tio n. The velocity of th e enzyma t ie react ion was d e termined by m eas llring the amount of prodllct, d ih ydropte roi c acid , t hat was form ed. Experimen tal de ta il s we re similar to t hose' reported hy Brown ( I ).

1{( 'tH ' 1 iOIl III i Xiii 1'(, ,,

:2 :1 -I

'1' .-\1\1.1': :.2. fr l'el '('I'8ihie illiliiJ itillll oJ I nlole sU l/lil l'sis "If -'/lifotiliazole.

( 'omJ!()IIe'lii omil IPd <illrill ,l!, fir~t ifl('lI ha l ioll Jl('r iod

;-;1I1J"aihia>l,li l' '\ Oil!'

;-;lIlfalhiamll', I' .\ 1\ 1'- ,\1 \

('OIllJlOlll 'lIl :Id<il'd :11' 11'1' fir~1 alld hd ol"l' i he .'('('1' 1)( I

inl'lIhai il)1I lH'ri"d

'\ 011('

,\, Ji ll' 1'-. \ 1 \ 11-.\ n

FolalP l'qlli I ' tl 1('11 I..;

I'ro<i II I'('d

J.l ~ ;l,S

;i . :2 li .-I 1. 1

" T lw p(,l'p:l.\":tl ilill of i.lI C ('('a(" lio l l Illi xl ll l"( ~~ :lll(ll h(, inl' lIl "llio ll 1'1I11l1ililll>, \\"('1"( ' a ~ ,Ic-,wrihcd in I h, ' i " xl. 01 hl' r d( ,tai l, III' l hi . .; l'X P(~ ('illwn l wprc a" dl'~ l"I"i l)('d hy B r"II'11 (I).

o 20 40 60 80 100

LENGTH OF FIRST INCUBATION , MINUTES Flc . 4. The effect of im:uhation time ( the first incuhation period ) on the irrcversible

inhibition by sulfathiazole. Durin g the first incubntioll period , reaction mixtmes con­tained sulfathiazole, 2 - amil}()- 4 - hydroxy-6- h yclroxv111 eth~' ldihydropteridin e, ATP, ~[gCl ~, and ell zyme preparation . After incubation at :37 0 for the tim es shown, p-AB was then added to each reactioll mixture and all were then reincubaled for 2 hollJ's. Enough p­Ail was added so that if the p-AB and sulfatlliazole had bee11 added at the S;,111 e tinl(' 11 0 illhihition h, ' slllfat hiazole wOllld han' hee ll ohsern'(l.

OIl this "i rreversihle inhihition" is g iven in Fig ure 4. As the incuhation timc ' is in ­c reased tIll' deg ree or irre versihle inhihition is also in creased . This sugges l'l'd that an l'Jlzy mal'i c reaction in vo lv in g su lfathiazol e as a sllhstrate mig ht he occ lllTing durin g the first incuba tion and that t hc Ill'! effect of thi s reaction is to either li se up other

suhstrate or to form a produl'! that a frects tlw forlllation of dih ydropteroak from p­AB . Furth( 'r l'X pCrilllc nts indica ted tha t' in ordn for the il1Tversible inhihition to oc­cur, ~ [ g ++, ATP and the clihydropt e rid ­inl' suhstrate had to hc prese llt il'. the r ('action mixture during the first in cubation p e riod . Thi s supported the v iew that a

Tlll enwlio/lal !ol/nlal lif L e}! /'o .,·!! 10()7

product was formcd alld sug)4('s[('cl that tIl<' prOoll ct was all a llalo)4 of dili ydroptcroat<' in which tlie p-)\B portion of this prodlll:t was replaccd hy sulfathiazole . One of the ('(feets of sll ch a pheilom cilon should he that the oihydropt{'ridillc suhstrate wOIild be COnSllI1wJ by rcaction with sillfathiazole and thll s not be avai lahle for reaction with p-AB after the Iatt cr compollnd was add C'c.l to the reaction mixtllrC'. I f this were till' case, thell hy incrC'asi llg th(' cone( 'ntTatioll nl the dihydroplcridille, one shOldc.l ohseJ"\"(, , l n' vcrsa l· of the inhibition . Sli ch all experi ­ment was dOlle and it was , indeed, fOllnd that high conce ntration s of the dihydrop­teridine in rcaction mixtllf"l's did ll'nc.l to rcverse the inhibition ( '1) .

Confirmation was ohtaincd [or the forma­tion of a slIlfa dmg-contailling product by the in cubation of sulfanili c acid-S :I :. with the cnzyme preparatioll , the dihydroptcr id­inc, ATP, and ~f g+ . Analysis of the incubatec.l reaction lI1ixtlire b y lI1ea ns of paper chrornatograph~r followed by radio­autography indica ted that a rad ioactive produot has been formed ( I ) . The forma ­tion of this product was shown to be de­pendent on the presence in t he react ion mixture of the dihydroptcridine, ATP and Mg++. This provides strong support for the view that sil lfa drugs a re ahle to re­place p -AB as sllbstralc for the enzyme. Thus the ohservcd competitive inhibition can properly he descrihed as a case of competition of su lfonamidcs with [1-A 13 as substra tes for the enzymc. Once the su lfa has competed stl ccess fllll y and become"s bound to the enzyme, it can th en rcad in the way that p-AB reacts to y ie ld a pro­duct.

Resistance to sulfonamidcs. Pato and Brown ( II ) have isolated a number of mutants of E , coli that will g row in the presence of a higher concentration of sul­fonamide than the parent wild-type strain will tolerate. An examination of these mll ­tants revea led that they can be divided into two classes. T he enzyme system ex­tractcd from cell s from one grollp of thcse mutants was identica l w ith that obtained from the p ,U"ent strain as rega rds scnsitivity to sulfonam ides and the !\f ichaelis constant

for p-:\I l. TIH'se ohs('J"\ ',l ti ons sll ggest th,\t th('s(' Illiitant s an: lc~s sCllsitiv(' to the grow th-in hihi tory propcrties or 511I fOIl,I ­miclcs by heing relative ly impernll'ahlc to the drllgs. The second class of mlltants eont a illed all enzym(' sys tem with a M ieha c­li s constant for p-A B .identical to that of thl' enzyme from the \\'ild-type stra in , hilt \\'as lcss scnsit i\"(' to t hl' in h ihi tory proper­ti es of the slilfon amid('s. Thll s, these 11111 -

tant s appeared to contain enzymcs a lt f'rt'cl to the ('xtclll that the comhi nation of th l' Clli'.yllll' wit h sl dfonamide is af[ected, Onl' sll ch mlltallt from this group contained an c llzyme so altered that it was not stahle to heating at ~6 'C , although the enzyme from the \\ 'ild-type strain was completely stahle to this treatment.

Since these mlltants were all isolated In' the multi step technic, it is like ly that th'e Illiltants that WlTC examined contained mon ' than n1l1 tation and that the observed phenotyp ic characteristics wcre therefore the res lll t of more than one Illll tatiolla I cvcnt. The chances 'secm good, for exam­ple, that those mutants that were observed to contain a ltered enzymes as a result of a mtltation or Illutations probably also might have super-imposed O il these mlltations others that might cause these organ isms to he relatively impermeable to sulfonam ides.

Wolf an d Hotchkiss ( ~I ) have ana lyzed a numher of mutants of pneumococci that arc res istant to sillfani lamide a.nd have re­ported that the enzymes from these mu­tan ts have a lower affi nity for the drug than the enzyme ex tracted from the wild­type sensitive st rain. They could End no evidence that altered permeability played any part in imparting resis tance to pnell ­mococci.

Other possible mechanisms that could theoretically be responsib le for resistance to sulfonamicks are: over-production b y the resistant cells of p -AB or ovcr-produetion of the p-A13-utilizing enzyme. T he only report that has appeared that sllpports e ithe r of these a ltematives in one hy Landy ct al. e') in w hich they claim that strains of Staphylococclls al/rellS res istant to 511l fona­mides ove r-produce p-AB.

3.'), -1 (PI. :2 )

SU~I\IAHY

TIll' l'llzymatic reactioll s that kad to tiJl' hiosynthcsi~ of tdral.ydrofolic acid are as follows:

( 1) 2-amino-4-hydrox~'-6- hydroxYl1lcthyl ­dihydro ptericline , p-:\ B .\ I I' dih~',

dropteroic acid P ) dihydroptcroic ~1C id -+ g llltall1ic ac id

A I \' dihnlrofolic acid ---> -

( :j ) dih\'(lrofolic acid 11' :-; 11 telrah\'(lro---->

foli c acid ,

T\\ 'o enzymcs ar(' ill\'()h ·ccl ill th e formation of dih~ 'dropttToic acid ( reaction 1 ) . These two hav(' I)('cn separated h~ ' chromat ogra­phic technic a nd it has hccn fOllnd that Olll' of these cnz~'nws ( Enzymc :\ ) catalyzes the formation of an intermediate From the ptl'ridine s llhstrate and ATP. The second cnzyme ( EnzYll1c 13 ) catal~' zl'S the l'OIl\ 'c r­sion of ihis intertlwdiate and p · :\B to c1ih y­dropll'roil' acid. The c nz~' lnat il' fOrJna t ion of dihydropteroic acid from p-.-\B and the ptcridint' intl'rnwdiate is inhihited hy slll ­fonamic\c s. Of a snic~ o[ s lIlfonamicks tcsled , s lIlfathi azolc ",as the most (,(kcti\'( ' illhibitor and slIlfanilamide thc least efrel' ­tiv t'. Expe rim e ntal ev iden ce has bcen o h ­tained that th e enzym e act llall y u scs thc s ldFon amides as substrates with till' forma ­tion of sl dfonamicle-containing procllH: tS that wOllld Ill' analogs Ill' dihydroptlToic acid. Thlls, the in hibitory propnties of s ld ­fonamide~ can he ascrihed to these com · pOll\lcls competi n g with p -AB a s suhs trates for the e n zyme. This differs Frolll the tradi ­tional view that sulfonamides arc c lassi c competitive inhihitors of the utilization of p-AB .

REFERENCES I. A:\(; ILH, H. B. , BO(lT II E, J. TT ., 1 fL'TLIII:\CS,

1\. L., \10\\, .\'1' , J. If. , St-;\ II1 , .L STOKSTAIl, E. L. n. , SI' lIlIo\Ho\\', Y., \\ ' ALLER, C. \\'. , COSL·I.ICIf, I) . B. , FAIIHE:\IJACH, \1. J. , Il pLT()UIST, \f. E., K Ul I, E. , l\'O HTIIEY,

E. If. , SEI': C EH , D . n., SICKLES, J. P. a nd S\IITIf, J. \1. , JH. The structure and SY II ­th('sis of tIle li V('r L. C{iSI ' i fadol' . ScicJl ('(' 103 ( 19/16 ) GG7-669.

2. BOCCIII EIH, S. and K o!''I', ll . Synthesis uf pteroic acid illtermetii ,ltcs frolll 2· amino­·1· h\ 'l 1 n)x~ · ·n- Il yd rox~ ' nl('t h~ ' 1 d ill Yllropte ri ­dill~ by S/aphljlococclls Of/rCIlS and l~scl/('ri('hia coli , Bact. Proe. (19G5) 7-1.

.l . BII()" .' , C. \1. l ' llpuhlis lwcl Oh~l'r\ · ;lIi()n s. ·t BIIO\" , C. \1. The hi os\ ntlil'sis of fo\i c

acid. II. Ilihihition h \' slIlfon:lIll ides . J. Bio]. ChclIl. 237 ( J962 ) 5:36· .">-10 .

,,) . Bl\o\\':\, C. \1. The S) nthcs is of fulic acid by ee11-free ex trad s or Escherichia co li . Fed. Pmc. 18 ( ]959 ) ] 9.

(i. BJlO\n:, C. \f. , \\'EIS\IA:\, H. A. and \ 10 1.­XA n , D . A. The bios~ ' nth('sis of fo\i c ac id . I: SlIh,tratl' :1I1l1 l'ofactor reqllin'llIl' llt s for ('))z\'matic s\ ntllcsis Il\' ('( ,1\ · fn'l' l'~ ' tra cts of ES('/If'ricflill ('(I Ii . r Ilin\. Chell!. 236 ( 196 1) 251-1·25-11.

, . CHIF!'I ", \1. .r. and Bl\o\\"", C. \1. TIll' bios), nt lH's is of foli c acid . III . EI17.) ma t ic formatio)) of dihydrofnli e acid from dill, ·· dropterni l' acid a lld of t('\r a h~ ' clropteroyl ­poh-glllt :llni e acid COlilPOllllcls from tetra­h Y'dr%lic acid. J. Bio]. C hf'lIl . 239 (J96-1) :3 10·:3 16.

R. J AE:\ ll KE . L. and ell \:\, P. C. Di(' Bio­s"lI th ese der Fol s:i llrl' . An ge\\' . (:1\('111. 72 ( 1 ~J(i() ) i.'i~- i5.l.

9 . L .\:\I)\ ', .\1 .. Lo\ II KL' \I , 'N. \\ '., O~\\,ALJ),

E. l a lld STHJo:I CIITOFF, F. IlI crcascd S \ ' II ­

t II ('s is of p ' :lmillohenzoic :lc ill assoc iat ed wit h t\1(' de\'(' lopmcnt of slllfol1amide re­sis t:lIlCl' in S/lIpIIU/OC()C(,lIs (J ll rI ·II S. Seil'llcc 97 ( I ~)-I:3 ) 265·267.

10. LASCELU. ~ , J. and \\ ' OODS, D . D . T h e S\'1I ­thes is of foli c acid h v B(Jc/ni lllll co li and S/lipliU/oC()CClI S 1I11r1' I/S and it s inhibition h \' slllpl,oll amit\es. Briti sh J. E:\per. P a th . 33 ( I ~).'52 ) 2kS·:3() 3.

II . \IITS ll IlA, 11. , SlIZI 'Kf , \ '., TADI ': IIO\, K. :l lId KA\\ ·.-\f, F. Biochemi c:l l s tlldi l's Oil pt e ri . dilles ill plalit S. Bioge nesis of folic acid ill g recli 1l':I\'('s: e ll z"lnatic synthesis of dih ydropte roi c ac id froJll g uan os ine COIll ­

pounds and mechanislII of it s s)11 th e ti c patl\\\· a~' . l "itamillo" 12 ( 1966) 192-JOe!.

1 2. :'\1~1~10· S .\IlTIf , n. n., L ' SCELLES, J. and \\ 'OOIlS, D . D . The S~ ' lItll cs i s of foli c acid 1)\, Slri 'p/(I/JIIl'lnilllll plllll/llrJlIIl alld it s illhihitioll h v slllpllOliamidl's. Briti sh J. Exp{'/'. Pat h. 29 ( IU-J8 ) 26-1·2S I.

1:3. OJITIZ , P . J. :111(1 I10TCIIKI SS, H. D. The 1'11/.)1I1:1 t il' S~ ' lIt hcs is of dih) 'drofo1at e and dih ydrnptl'roatl' ill ('(, II -free prc'para tio ll s from wi l!l-t \' pe :lIld ~ lIlf()lIalllid e · rcs i s t :ln t

p"(' lImOl·O('CIiS. 11 ioc\il' III ist ry ( \ Vash . ) 5 ( IUGG ) ()'j - i -l

1·1. " .\TO, \1. L. a llcl l\H()\\':\, C. \1. ~lccll:l ' lIisms of re~iqal1ec of Escllrri('liia coli to s lllfol1:1ll1ides. Arch . Biocheill . & Biophys. 103 ( 19(·l.)) 4-t:3- ~-I 8.

,588 lll/ erl/a /i olla l JO/lrl/al of LI' IJ roS!/ JH67

I,). S IIH rJ \ , T. I': II Z\ Illi e s ylll l H'~i~ of lo lil' avid lik(' (,OIIlPO llllds by ('c ll - fre{' l'\lrads 01 LlIl'/ o -l)(Jcillll s 1II'11IIiI/OSIIS. A rc h . IliodwlIl. & lIi ophys. 80 ( 19S!)) 1.'5.'5- 1 (i I.

I (i . S IlI OTA , T. all cl DI SH \ I': !.)' , \\. 0:. Til l' (',, ­

z~ ' lIlil' s)'1I 11ll'sb of clill\'clrofolal e frolll

2- a 111 i 11 0 - -I-h yclroxy - fi - h ~ 'd !'OxYlIlei h yld ihy

droplcriclille ;111 ( [ p -a mill o - \J e ll /,()y lg llli a

m al<' 1)\' e:drat'ls of L(fc lo- II(f(' illll s p/(fl/ ­

l(frl/II/. IlicwlH'lll . Ilioph\·s. Ada 52 ( I ~J(i I )

·1(i7 -47:\.

1-:- S III OT ,\ , '1' ., DI SI!,\ I'; !.)', \\. :'\. alld .\1 <. C: '\Y'':; \1. P. I'reparal io ll of dilrydropleri­

cli"ediphmplral(" a ll illlerllH'clial!' ill dihy­clmlolal( ' s~ ' 1I11H'sis. lIiodH'''!. lIiophys.

Hl's. Co III III II II. 7 ( I D(i:2 ) I !J4 - IUH. IH. SIII(),!' ,\ , '1'. , DI SH ,\ I·:r.)' , \\. 0:. a "d \ 1(. ­

CA:'\:'\, .\1 . P. T h e e ll z)' llw lic s~' lllhes i s of fo lal e- like cOl!lpoII!l(l s from h \'dro.\y­

IlIclh~' ldih~ dropleridille [1yroph o~pha t c. J. Bio I. C Ir !'m . 239 ( I ~)(j -I) :22.'5D-:22fi(i.

19. STOKS'!"\II, E. L. n. , I I I'TCiII '!.S, B. L. , '\IO\\,AT, .I . II. , I\m)'!' 111-:, J. 11. , \\' \LLElI ,

C. \\ '., "'(. 11 ': 11 , 1\. II ., S I·: \II1 , J. ;I lld S I ' 1I

Il.d {()\\', Y. 'I'll!' degr;r d a t inll 01 llr!' fcmlt'l! ­[alioll L(fclol)(J(' il/lIs C(fse ; I ador . .I . Aill ni ­va ll C IH'Ill. SOl'. 70 ( 194<'1 ) .'5 -D.

20. \\'EI S\I ;\:'\, H. A. a ll d I\Ho\\ ':'\, C .. \\. Th!' b i ()s~ ' IIll j('sis of foli c acid. \ ' . Charader­is lics o f the l' II zyl!H' sys lem lhat l'a l a l ~ ' zl's t h e sn it h cs is of dihl'llropll'roi c acid . .I . lIio !. C hc lll . 239 ( f !)(i-l ) :32(i-TI I.

21. \\ '(lIY, II . a lld 1I 0TC IIKI SS, n. I) . (;ellcli ­('a Il l' mod i fiecl fol ic acid sy ll t hes iz i fI g (' II '

/'~ · l!l es of P"(" "1l 0CO(,(, II S. lIi odl!'m i s lr~ ' ( \\'ash . ) 2 ( f !)():3 ) 1-I .'5 - t.,)O.

.).) \\ 'OOIlS, D . D. Th!' n,lalin" 01 p ' al llill() ­IlI'li zo il' avid to lh!' l!H'vh a lli slll 01 lh(, av­l ioll of s ulpiiallilaillidc. IIriti sh .I . E .\ p('f' . Palh . 21 ( ID-fO ) 7-' -DO.

2:3. W OODS , I). I) . \ lelalJolil' re laliolls b('­lW('('1! p-amil1olwnzoic :lcid an d foli c acid ill microorganisms. III C. E. \\ '. \\'o ls IDII ­IIDlm e and \1. P. Callwroll , Eds. , Ciha ' ,'o llllda lio" S\ 'mposillm o n C h em is try ;lIld I\io log,· of Pt cr idill(, ~ . I\os loll , Lillie, Bro\\,n' an d Co. , I ~).') -I , pp. 220·2:3fi .

DISCUSSION

Dr, Prabhakaran . 1 n s tl\(li ('s of this typ e it is cO ll1mo n practi cc to d o Lincwca \'lT­Bllrkc pl o ts o f thc \'(' sults. YOIII' data did not s how a n y. I (;1\'(' ~ ' O ll d o n c so')

Dr. Brown. \ \'c ha\ 'e done so on the ef­fect of thc inhihitors on the initi a l \'elocity of the (' nzY lll e.

D,·. Prahhakaran. The pathways of hio­svnth esis of r ed ll ced forll1s of foli c acid I{a\,(' h(, (, 11 \\'c ll wo rke d out in othlT mi ­croorgani sm s a nd ill a nimal tissues. Do YOII find a nythin g n ell' or difrcre nt in E. col i!

Dr. Brow n. 'I'll(' pathways \\'('1'(' the same.

Dr, ~Iorrison. I \\ 'ondn if YOII ha\'(' any kint'li(' infonnatioll on tIl(' \Ii c haeli s ('011 -

stallt for p -A B as compa re d with sulfa thia ­zo ic whcn the lattt'r fllnction s as a substrat!' for Enz\'m (' 13 , the condens ing ('nzyme.

Dr. Brown. TIl(' K, for p - .-\ 13 has I)l'('n d etcrmin cd to he approx ill1 ate l~ ' l.O x 10 c' },,1.

No cxact \'a lll e for th c K, for sulfa thi a ­zo le has bec n dclcrmillcd , althollgh \\ 'e do kllOW that the latt (,r (,O IllPOlilld is lltili zed more cfrcdi\'(' ly t h an ]1-. \13 as sllhstrak .

Dr. Bushhy. Dr. Brown has sh o\\ 'n liS thc s tage at which s ili fonamides a ct in thc synthes is of the co-cnzym e, te trahydrofoli c acid , a nd w e shall la ter rc f('l' to th c potenti · a l ill g effect o f p yrillll'l h a millc and trime ­tlwprilll Oil Ihe antihactl'l' ia l a lld a llti­malarial acli\' ity of dapso ne. I s h Ol tld likc to take thi s opportU llit \' to point ()lit th e s tage in thi s synthesis in w hi c h p y riIlH'­Iha min!' alld trilllt't h opri m ad. Th!'y int!'r­fcre with tIl(' COIl\ '('J'sio n of dihnlrofo]at!' to il s ll'trahydro form Ihroll g l; comhillin g \\'it h tctrahydrofo la tl' reductase. The po-1(, lIli atio ll is there fore dill' to the SlIlfolla ­Ill id ('s a lld th l's!' p y rimidin('s act ill g ill tlf(' sa m c hioch e illi ca l p at ll\\'ay hil t a t difklTIlI s tagcs.

,~", «( I)t <J ) ' )') , "-t _ . .;...J 3S9

Dr. Prahhakaran , Is it not trll P th at the d C(' ITascd to OIl C th a t prodll l'l ,d tIl!' sa llH' ,' Itlfolla mides di/k r fro II I the s ltlfo llt's ill hlood le \ 'c ls o f dm ).!:, DDS \I 'as foulld to 1)(' that tlw\ ' do !lot C;1 II.,(' ;1IH'lI1ia ;lIlcl a re Il o t 110 lI1 0 re he m() ly ti c tha n s ill f() ll ;lm icl e", dledi\ 'I' ill l e p rosy'~

Dr. Brown. T \\'o ltld han' to ask D r. She pa rd to a llS\I'c r th a t qll es t iOll ,

Dr. Shepard. llliti a ll~ ' the impress ion w as gaill ed that DDS \\'a s II1ll ch Illore toxic thall tlH' s lllfollall1ilks with respect to the prodllction of Ilw tlll'll1og lol>ine ll1i a a III I ancmia , Latl ' r it was karncd that the dif­icrence lay in tIll' mll ('h s lowe r ('xcrt'tio n o f DDS , :\ s a conseqlt('n lT, w hl' ll DDS w as g ive n in the sa me dail y d osage as slllfona­ll1ides a vc ry hig h hlood k w l was hllilt lip , ",ith the produ ct ion of sl've re he lllol y ti c rea ctions, I I ()\\ '\' \ 'e r, \I 'he ll t hl' dosage \I 'as

DI'. Bees. \ Vh a t expn ie nl'l' h al'(' yO Il h ad on the e n1l' rgen ce of resis tan ce to DDS, and othe l' sulfonamides , to strai ns o f h ac­te ri a o the r thall m ycohacte ri a:) \Vh a t was the p :tttl'l'n of res ista nce, an d , in partic ltl a r, \\'e re the l'( ' eX:llnpl l's o f "s ing ll' -s tT p 11111 -

t-a llts ,>0)

Dr. Brown. \Ve h:1l'e (IolH' 110 work with DDS as an inhihit or. The II1ltt a ll ts that \IT hal'e exa min ed tha t a rc ]'( 's is tant to sllifona­mides have a ll h Cl' n d e ri n 'd from K coli , and w cre ohta ined as ll1ulti -s lTp nlllt:lIl ts, The resistance p a ttc ll1 s that WI' have oh ­served a rc (k scrihed in the text.