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Studies on the β-galactosidase isolated from Escherichia coli ML 308. I. The effect of some ions on enzymic activity
Studies ML
on 308.
the
fi-Galactosidase
I. The
Effect
of
isolated Some
Ions
from on
Escherichia
Enzymic
co/i
Activity’
The eftects of magnesium, sodinm Ltnd potassinm ions on the activit), of P-&tctosid:tse have been stndird in tletG1. It has been found that the ionic effects vary with snhstrate. When ort,ho nitrophellyl &wgctl:rctositle is the snbstlate both magnesium :tntl sodirlm ions must be present, 1o obt.ain the maximum r:ktRIyt,ie rate. When I:tctosr is the sllbstratc pot:wsium ion is most eHectivc. INTR( )I)UCTIO?; 111 a previous
that’ magnesium
paper
(I)
it) was reported
ion n-as required
for the
f’at alytict wt ivky r)f p-galactosidnse prepared iI, this laboratory from E. co/i MT, 308.
Wallenfels rt ccl. (2) found 1hat a highly purified preparation deriving from hII, 309 did not require R4g ++. It, n-as reported that Xl++ n-as used ill the mzyrnc assay during purific:ation (2, :‘,) but, no \)i\-slc~lit~ ioil \vas prwiwt, in tlie assay system used for the purified preparations. Ledcrberg (1) ohscrvcd no cff’wt~ of hiralt~lt cations upon the ac*t i\-it y of crude extracts of st~raiii K-l 2. I”rorn this same strain, Tiuhy and Lardy (5) prepared purified &al:wtosidase alid reported that “Mg++ and >,II~++ actil*at e slightly ill a system fully wtivated hy AYa+.” 111 a rcwnl review C’ohn stat cd (6) that, “p-galact osidases show hivalcnt ioll (Mg++, IbIn++, I~(~++) act ivatioii, which is masked ill t,hc:L. co/i cilxyinc riiitil it, is treated \vith L This invrstig:tl ion K:I~ snl)portetl in lx~rl It) rcwnwh grant h-1581 from the S:~tjon,l Institntc of Ar.tllritis :tntl 1Ietnlmlic 1)isr:wx of the ?;atiord Instit utcs of Hwlth. l’rl\)lic Hcnlth Srrviw, :~,ndin pnrt I)\- gr:~nt SSF C;-2308from the N:itionnl Science Formtl~tion. I’resentrtl in Ixirt, at, the April I!%0 meeting of the Fctlrr:tiiori of Amwic:tn Rocicties for ICsprrimental 13iology, ClliPqqo. ? S:lll)l)ortcltl in p:wt 1)~training grant 2A-14(I’B) from the S:ltion:Ll Invtitnte of Arthritis :nltl llct:ll)olic IXscascs.
cornplexing agents, after whirh a specific effect of nlg++ (and to a lesser extent of AIn++) is revealed.” TSarher Cohn and Monod (7) had reported that Rig++ a&vat et1 slightly, and Mu++ inhibited the action of &qdwtosidasc on lactose. ILewetly, Itickcwherg (8) found that R!In++ protwtcd against dilution innctiratiou of p-galactosidaw (7) hut, that ,14g++ was not cclui\-aleiit. l’ho striking cffwt of uili\xicilt. ions 011 the c~atnlytic~ acativity of p-galactosidasc was
first uot ed by Ledcrberg (4) who employed 0NI’CP as a substrate in a simplified assay procedure. The prcwnw of Sa+ vxs found colltlucti\-e to maximum ratrs of hydrolysis. The cffec%s of \xrying X:r+ ~ollc.eilt.ratiolIs oxwa suhstnntial pH range was documented by Cohen-Bazire and Monad (!)). Tiuhy a~1 lmdy (.5) st atcd that, tht cffwt of Na+ was iioiivnriailt~ with suhstratc type, but, Cohn awl XIo~~otl (7) otwrrcd I\+, rather tha11 Na+, to promott, :t greater ratv of hydrolysis \vtici~ lactow TV:ISthe sutwtratv. .I lxicf discussion of this puzzling situation has breii recwrdcd (IO) aud has received cvmnwnt in a rcwnt rw-it:\v (1 1). The spwulatiw~ I hat,
271
the enzymes from strains Ii-12 and MI, :508 were subtly differelit (5) was at varinllw with other evidence which illdicuted that, all ,&galnctosiduscs from E. coli mutants were indistinguishahlc (6). Much of t’he work referred to ahovc utilized ext,racts or purified preparations whose homogeneity could not be \wified. It seemed worth while to reinvestig&e these ion efftcts wit,h enzyme believed to he homogeneous. MATERIAI,S
ANI>
RlETHOI)S
The galnctosidnsc prrparat ions used in this investigation were prepared from EG.coli hII, 308 1)~ Dr. Dexter Rogers and from strain K-12 by l\ziss bI:iili Hrinsoo by a method previously descrihrd (1) Highly purified prepmxtions of hovine mam mary gland Klucosc-li-phospll:tte drhq-drogenase were kindly provided I)!, Air. Gordon .Julian. A partially purified hcxokinase (Type IV) M BS geueror~sly supplictl by the Sigma Chemical Company, Ortho nit.rophenyl p-n-galactoside \r>ts purchased from Sigma Chemical Co. lactose (c.1’. grade) \~as treated with ion-exchange resins to remove trnres of inorganic ions and crystallized from :rq,wwus ulcohol. lIethy p-u-gnl:lrtositlr W:IS prepared 1)~ st:ind:ad methods. The pH stat rlsed was :I 1%Funct,ion:tl Rccortling Titrator purchased from the International Instrument Company, Cmyon, California. The polnrimeter used ww a photoelectric model m:tnuf:tctured hy t.he Rudolph I’olxrimetcr Co. :tnd made :tvnilnl)le through the rourtwy of I)r. Johrl Schrll,n:\n
The experiments whose data are summarized in Figs. 1 rind 2 were carried out, as follo\vs. Ewh incubation had a total volume of 1.00 ml. and ~OJItniued 2.65 X 1Om3mg. rnzymc. The suhstratr: oNl’G, was present in :I concentration of 10m3 M. The buffers were 0.05 df TrispTht:. 1ncuh:ttion was for 10 min. at 25”. The hydrolysis of oKI% was estimat,ed by adding to t,hc 1.00.ml. incubntion smnples 0.4 ml. of M ?ja&O:, and measuring the optical density at 420 *np (4). Optical density or nbsorb atice units per IO-min. assay relmrted in tal)les or figurrs can hc converted int,o mpM/min. I,,v using a conversion factor. Thlls 21.7 m~df/min. corrcspends to 1.0 optical density unit. Tris-ThG I)utfers containing substrate were prepared as follows. Thioylj-colic acid, 0.05 ‘11, was m:ltle up in oxygen-free w:tt,er. To 10-m]. s:rml)les were added 50 ~1. oM’(: in pyridine (60 mg./ml.); solid nnalytir:tl gratlc Tris was added t 0 :i pH ne31 that desired, and the final pH was adjusted wit,11 a pII-stat. The titration w-as donr in :I nitrogen atmosphere, and solutions lucre stored t~ndcr nit rogcn.
The method used to meas,Ire the esteut. of 1:~ tow hydrolysi:, was ii spect rophotomet ric* enzymic rst,im:ation of the glu~osr 1”odlwctl. The eriz,vmic rcagent~ \v:*s prep:trcd 1)~ adding to 18.0 ml. of 0.05 If ‘l’riswrwt at r t)utirr, pH 7.0, 0.2 ml, of M hJg(:ll! , 0.4 ml. highly purifictl glllcose~(i-phosphate tlrhydrogenxw (1.7 mg./ml.), 0.4 ml. ‘l’l’r (20 mg./ml.). 0.4 III]. hrxokimrse (Sigma ‘I’ypr IV, 10 mg./ml. J :m(l 0.2 ml. .4T1’ (200 mg./ml.). Aliquots of 0.98 ml. bvere t em1~cr:~t~~rc-cc~r~ilil~r:it cd 31. ‘15” iu the silica ruvet t,c lwfore ILSC. This wagcut rollltl IN: stored 31 - 10” :intl thawed repeatedly bkithorit iqprecGal)lr tlrteriorntion alt bough long standing c:tusctl iniwt ivation. Addition of lo--’ moles glrirosc (5 ~1. of 0.0 :II glucose) caused :I rapid inrre;rse ill optical density :rt 310 mp as mc:tsurcd iu the ISwkman I)17 qwt rophot~onicter equipped with “thrrmo-spacers ” to m:rint:Gn a 1empcr:tture of 25”. The tot al A:iJ,, for t.his :miount of glucose was O.WO opt~iral density unit in less thari 10 min. Sinyr >Ig++ \V:LS rrquitwl t)y the glucose tlet,cction syst cm, it ~viis newss:try to inrut)at.c 1)uffcred lactose wmplcs with the enzyme in small t utws, to stop the rraction 1)~ pl~~nging the tribe in boiling \v:tter for :I-5 min. , and, :tftrr cooling, to unalyzc appropriatr :diquots. Aliquot size was chosen so that) first -order kinetics obtained in the glucose tlrt,ection system. I,nct ose solrLt,ioris of v:wying ronccnt,ratiofi were prepnrctl by diluting a cwnrcritr:ttrd stock solution of 1:tctosc bvitli O.OY M ‘I’riswtcrtntr hllfler, pII i.0. To :t 0.2S1nl. sample of such a solution iu :I smxll t ulw was atldcd 5 ~1. of 0.5 dl ?;:tCl or KC1, :m(l thr tulw W:LSrqlGlibr:rted at 25”. To this was uddetl r:ipidly ant1 with good mixing 20 ~1. enzyme sollltiori (0.132 mg), After 2.0 miu. the incuhntion tube ~v:ts plrulgctl into boiling wat,er to st,op rr~zymc xction :LII(~ then I)rought, to 25’. This \V:LS done in a staritl:ndized bv:iv so as t 0 minimize scvrlnl variewrre :~nalyzed ties of esperimcrit:11 error. _4liyuots ~~~r~tro1~l~otomctricall~as indicutrd prcviourly.
I~:xpwimcnts to dcterminc~ the approximate concentration of Mg++ needed to ohtail1 maximum hydrolysis rates arc exemplitied 1)~ IJig. 1. In the next set of experiments, summurized ill l’ig. 2, the cwnwntrations of both ,I’a+ and Mg++ were vnricd. It) may tw noted that, R’Ig++ is far more effective at low pH values. Further, it can be seen that the optimal pEI in the presence of ndequatc amounts of both ions, Sa+ and TV@++,XLS
27:;
\
\ \ 0 \
NoMg++od,jed
-----------v-l--
3
2
0
4
5
- log I Mg++l
0.60.5--
/
0 /
/o-. 0,
\
/ a? z 0.4-a g 0.3--
/
0.
0 /
‘\
, / /
\
AC0 ++o++
\ 0, O*
4.
/ / P’
0.2-m
/ 0. I ~~ /’
xxx *
*
,‘. Y ,“-
6.0
kii
,o-
*O
-o-.-o-.
3’
** o+
5.6
,‘\
OkX
%’
/
,O’ /
040’ 5.2
/
x x
o6.4
*’
+;o
0
-o-.--
-A’
6.6
>’
--0 7.2
7.6
6.0
6.4
PH
ti.8, hut at ION vnlucs of either, the pI1 optimum n-as raised to about 7.1. These data cmfim and extend those of Ref. (0). 15sw~tially the same values \vere ohbained with trkncetntr buffers a.s UWT ohtainrd
with tris-ThG huffcrs. This indknt ed that ThC; was 11ot inducing a11 Aig++ efkrt 1)~ cornplcx formntiml 1101’Tv:is it inlpnrt~nut, during the short. iuculmtiou period, to t alw rneasurcs iu order t 0 protect --SII groups.
However, stock enzyme solutions were always diluted with tris-ThG which had been prepared, and stored, in nitrogen. Another set, of experiments was done to establish a relation between Mg++ and Mn++ effects in such systems. n-0 essential difference could be detected bet’ween the rat,es attained in t,he presence of iYa+ and Mg++ and those attained in the presence of Sa+ and Mn++. The dat’a in Table I show the concentraTABLE THE
IXHIBITIOX
I
OF oXPG BY EDTA
HYDROLYSIS
IncubaGons contained IO+ 111oNPG Tris-acet,ate pH 7.0 and 1OW M Xa+. added.
in 0.05 M No hIg++
Optical density
EDTA M
,035 ,071 .200 .200
10-a 10-d 10-s 10-G
TABLE
THF, EFFECT HPI~OLYSIS
II
RIWWTIOS OF EDTA &GALACTOSIDASE
INHIBITION BY Na+
iTa+
OF
Optical density
.lf
,122 ,092 ,035
10-1
10-z 10-3
Incubations contained 1OW M oNPG in 0.05 M Tris-acetate pH 7.0 containing lo-’ M EDTA. TABLE REMOVAL OF EDT-4 p-GALAC,POSIDASE Mg++
III INHIBITION BY h3g++
OF
Optical density .~______.
__
T--
10-Q 10-s 10-4 10-S 10-o Ko EDTB
tion of EDTA which will cause inhibition. In the next experiment,, Table II, it will he seen t’hat raising the xa+ concentration tends to reverse EDTA inhibition slight,ly. In Table III t’he dat,a clearly illust,rate t,hat Mg++ removes EDTA inhibition and is consonaut, with t)he evidence of Fig. 2. Since the amount of enzyme present n-as identical in each incubation of t,his series, it, is evident t~hnt Mg++ does more than activate slightly even when adequate Iia+ is present. Figure 3 shows t’hat the maximum \pelocity of hydrolysis in t,he presence of Mg++ and Sa+ was no great’er t’han in the presence of Na+ alone, alt’hough the value of K, was substjantially reduced. The value of Tin,:,, in the presence of K+ was 2 X 1OP moles/ml./ min.; in the presence of Sa+, -l.% X 10Py. The values of K, were as follows: K+, 3.:3 X lo-” 111; Na+, 10-3; Na+ + Mg++, 3.7 X 10-d.
0.460 0.458 0.424 0.090 0.075 0.450
Incubations contained 1OW M o??PG in 0.05 M Tris-se&ate pH 7.0 containing 1OF M Xa+ and lo-’ M EDTA.
OF
10~s OF
ox
THE
LACTOSE:
With lactose as a substrate, no Xr[g++ requirement was observed. In t,he presence of 0.02 J1 K+ or n-a+, the presence of 1 X lop4 AT EDTA had no effect. In 3.3 X lo-” ill EDT‘4 and no added univalent ions, t’he enzymic acbivit’y was greater than 10 % of Ohe maximum rate observed in the presence of 0.02 JI li[+. Figure -2 shows that in relatively high concemrations of lactose the presence of K+ allowed a greater velocity of hydrolysis than Na+. In dilute solmions t,he difference was less marked. Indeed, there was appreciable act’ivity (about’ 30 % of maximum) when no ions were added. Values obtained by extrapolation of the points plot’ted in Fig. 4 were as follows. In the presence of ii-a+: K, , 3 X 1OF 111; l;‘,n,, , 7.8 X lO-7 moles/mm. In the presence of K+: Ki,, , 7.7 X 10-3; I’,,, , 1.7 X low. The availability of a photoelectric spectropolarimeter encouraged us to study enzymic lactose hydrolysis by measuring the changes in rotat’ion. The rotation of 0.1 ~11 lactose at 25” was easily and reproducibly measured to 0.002” (X13 mp). When hydrolysis was effected with enzyme, only
p-GALACTOSIDASE
900 600 c
-3
-2
-I
6
I
2
3
4
5
6
7
6
9
IO
rx lo-3hA ISI FIG. 3. l/71 versus l/S plot of oiYP(: centration of each ion 10-S M.
18
hpdrol,vsis
rates in various
ionic environments
at, pH 7.0. Con-
t
8 6
0 FIG. 4. l/a versus trntion 10e2 31.
40
80
l/S plot of lactose
120 hydrolysis
160 cont,rasting
200 effect
of Nnf
240
"WlU
and of I<+. Ionic
concew
276
ItEITHEL
slow changes were observable. Apparently (a!,@)-lactose, (CY,/3)-glucose, and P-galactose all have rotations which are nearly identical. Further, at, pH 7 the P,CY mutarot)ation of galactose is slow. Thus, it, seemed advisable t,o abandon this simple physical method in studies with lact,ose, but it was used wit)h success in examining anot,her suhstrat’e. Had cr-gnlactose been liberated, rotst,ional change should have been observable. The results suggest that the fl-anomcr is released, but, further careful experimentation will be required t,o establish this.
Since &galact,osidase action differed so greatly in ionic requirements in the foregoing st,udies, ot,her substrates were examined. Methyl p-n-galactoside was chosen because it, exhibits a low rotaCon but would yield upon hydrolysis a product of much great,er rotat,ion. Initial rates of change of rotation were determined so that t,he rate would be relatively unaffert)ed by p,cr mut’arot,ation. Observations mere made at 365 rnp and 25”. As in the case of lact’ose, no effect of Mg++ was observed. In experimental mixtures containing 0.1 111 substrate in 5 x lop4 111 sodillm phosphat,e buffer, pH 6.8 and 0.15 mg. enzyme/ml., t,he rates observed were slightly greater than in the corresponding potassium phosphate buffer. In tris-acet,at,e buffer, where no inorganic ions were added, t)he rate of change of rotation was almost nil. When 0.1 ~11 subst.rate in 0.05 -11 tris-acetat,e, pH 7.0 was used, and ions added as the chloride at lo-’ ill concentration, t,he rates in the mixtures containing IX+ were slightly greater than those containing Sa+. DISCCSSION
The evidence reported in t,his paper indicates that, (a) magnesium ion is necessary as an adjuart, or roenzyme only in the rapid hydrolysis of oXPG, (b) it reduces t’he K, value, and (c) it, does not raise I’,,, in the presence of adequate ?;a+. It follows t’hat neit,her Mg++ nor Mn++ can be considered as an essemial part, of t#his enzyme system nor can they be considered act,ivators for all
AND
KIM
subst’rates. In no experiment were we able to demonstrate any difference bet,ween the a&on of NIg++ and that of ,&In++. Further experimentation will be required t,o discover whet her Mg++ can be excluded vigorously from the enzyme wit,hout, loss of activity. et al. have stated [(I I), p. As MaImstrom 13-l] : “another t’ype of artifact. becomes more common wit,h highly purified enzymes, namely, that due to t,he metal ions stabilizing the protein in dilute solution.” Since the use of oI%PG as substrate allows assays at very low enzyme concentrations, it. must) be determined whether t,he NIg++ effect is a function of the protein csoncentration for this sub&ate. Apparent,ly previous workers have not excluded all NIg++ (or Mn++) since the pH at which maximum hydrolysis rates have been observed is more often recorded as 7. Figure 2 shows clearly that, absence of n!Ig++ can be not,ed by a decided increase in t,he pH value at, maximal act,ivity. It is apparent that :I small amount, of Mg++ is more effective at low pH values than at high pH values. This would speak against competition by protons for complexing. l’rcsumahly this effect is related to the pK’ of some group on the enzyme. Ion-binding studies are planned in order to oht,ain more evidence. The observations made on univalent ion effcrt,s c~onfirm the claims of Cohn and Monad (7) and may be useful in explaining some contradictions. It is obvious that. the relative effwt,s of Ka+ and Ii+ depend on the concentrations of t,hese ions as well as hydrogen ion. Kot enough data have been collected to make evident the relation bctween substrate and ion requirement. In a recent#ly report,ed case (12), Kf was sho\vn to have a greater effect’ on the K, \.alue than on l’,,;,, . It is evident that’ t’his cannot, be the case for p-galactosidase when lactose is the substrate, but) it may be true for ot,hrr subst,ances. It is tempt,ing to postulate t,hat. K+ or Sa+ can be bound in such a way as to diminish hydrogen bonding, which may comribute to the st,abilit,y of t)hc CC zyme-substrate complex. Stated alternatively, some dissociable group at the “a(Ative site” may funct8ion more cfficient~ly in the presence of an ion contribut>ing less to reso-
wuw stabilit’y. This hypothesis is susceptiMc to experimental testing a11d may (wlitrihute sometjhing to an understancl& of unixdent iou effwts.
Proteins” (Benesch et al., eds.). pp. “Bi-41. Academic Press, Sew York, 1059. 4. 11~~~:~~~~~, J., J. fkckrid. 60, 381 (1950). <5 I~CBY, s. il. BNI) LARl>Y. H. iz., J. .1ta. C’hou. sot. 75, 890 (1953). 0. 7. 8. 0
1, Hu, 2%. $. I,., WOIXE, R. (;., ANU J., .li~zh. Biochcnr. Nioph?/s. 81, 2. WALLESFELS, Ii., ZARSITX, 11. I,., BESI)ER, H.. ANLI KWER, RI., 331, ‘I50 (1959). :<. \li.41,1,ENFEI,S, Ii., iTI “Symposium
&ITlIEL,
F.
500 (1950). LAI-LE, (:., Niochew. %. on Sulfur
in
COFIES-BAZIRE, (+., .4s1) iLIosol), J., f’orupl. w/id. 232, 1515 (1951). I)., asI, (;LASS, B., “The i\lrchIO. ~~~:LRc)Y,W. anism of Enz,xxw Action,” pp. “87-00. Johns Hopkins l’rws, lMtimow, 1954. 13. (i., ~NI) ROSENBERG, A., :I& 11. &1AI,MSTRihl, uunres in Enzyrrcol. 21, 153 (1959). 12. 'I‘AHOR, H., AND WYN(:ARUEN. I,., ,I. h'id. (‘hew. 234, 1812 i1050).
on 308.
the
fi-Galactosidase
I. The
Effect
of
isolated Some
Ions
from on
Escherichia
Enzymic
co/i
Activity’
The eftects of magnesium, sodinm Ltnd potassinm ions on the activit), of P-&tctosid:tse have been stndird in tletG1. It has been found that the ionic effects vary with snhstrate. When ort,ho nitrophellyl &wgctl:rctositle is the snbstlate both magnesium :tntl sodirlm ions must be present, 1o obt.ain the maximum r:ktRIyt,ie rate. When I:tctosr is the sllbstratc pot:wsium ion is most eHectivc. INTR( )I)UCTIO?; 111 a previous
that’ magnesium
paper
(I)
it) was reported
ion n-as required
for the
f’at alytict wt ivky r)f p-galactosidnse prepared iI, this laboratory from E. co/i MT, 308.
Wallenfels rt ccl. (2) found 1hat a highly purified preparation deriving from hII, 309 did not require R4g ++. It, n-as reported that Xl++ n-as used ill the mzyrnc assay during purific:ation (2, :‘,) but, no \)i\-slc~lit~ ioil \vas prwiwt, in tlie assay system used for the purified preparations. Ledcrberg (1) ohscrvcd no cff’wt~ of hiralt~lt cations upon the ac*t i\-it y of crude extracts of st~raiii K-l 2. I”rorn this same strain, Tiuhy and Lardy (5) prepared purified &al:wtosidase alid reported that “Mg++ and >,II~++ actil*at e slightly ill a system fully wtivated hy AYa+.” 111 a rcwnl review C’ohn stat cd (6) that, “p-galact osidases show hivalcnt ioll (Mg++, IbIn++, I~(~++) act ivatioii, which is masked ill t,hc:L. co/i cilxyinc riiitil it, is treated \vith L This invrstig:tl ion K:I~ snl)portetl in lx~rl It) rcwnwh grant h-1581 from the S:~tjon,l Institntc of Ar.tllritis :tntl 1Ietnlmlic 1)isr:wx of the ?;atiord Instit utcs of Hwlth. l’rl\)lic Hcnlth Srrviw, :~,ndin pnrt I)\- gr:~nt SSF C;-2308from the N:itionnl Science Formtl~tion. I’resentrtl in Ixirt, at, the April I!%0 meeting of the Fctlrr:tiiori of Amwic:tn Rocicties for ICsprrimental 13iology, ClliPqqo. ? S:lll)l)ortcltl in p:wt 1)~training grant 2A-14(I’B) from the S:ltion:Ll Invtitnte of Arthritis :nltl llct:ll)olic IXscascs.
cornplexing agents, after whirh a specific effect of nlg++ (and to a lesser extent of AIn++) is revealed.” TSarher Cohn and Monod (7) had reported that Rig++ a&vat et1 slightly, and Mu++ inhibited the action of &qdwtosidasc on lactose. ILewetly, Itickcwherg (8) found that R!In++ protwtcd against dilution innctiratiou of p-galactosidaw (7) hut, that ,14g++ was not cclui\-aleiit. l’ho striking cffwt of uili\xicilt. ions 011 the c~atnlytic~ acativity of p-galactosidasc was
first uot ed by Ledcrberg (4) who employed 0NI’CP as a substrate in a simplified assay procedure. The prcwnw of Sa+ vxs found colltlucti\-e to maximum ratrs of hydrolysis. The cffec%s of \xrying X:r+ ~ollc.eilt.ratiolIs oxwa suhstnntial pH range was documented by Cohen-Bazire and Monad (!)). Tiuhy a~1 lmdy (.5) st atcd that, tht cffwt of Na+ was iioiivnriailt~ with suhstratc type, but, Cohn awl XIo~~otl (7) otwrrcd I\+, rather tha11 Na+, to promott, :t greater ratv of hydrolysis \vtici~ lactow TV:ISthe sutwtratv. .I lxicf discussion of this puzzling situation has breii recwrdcd (IO) aud has received cvmnwnt in a rcwnt rw-it:\v (1 1). The spwulatiw~ I hat,
271
the enzymes from strains Ii-12 and MI, :508 were subtly differelit (5) was at varinllw with other evidence which illdicuted that, all ,&galnctosiduscs from E. coli mutants were indistinguishahlc (6). Much of t’he work referred to ahovc utilized ext,racts or purified preparations whose homogeneity could not be \wified. It seemed worth while to reinvestig&e these ion efftcts wit,h enzyme believed to he homogeneous. MATERIAI,S
ANI>
RlETHOI)S
The galnctosidnsc prrparat ions used in this investigation were prepared from EG.coli hII, 308 1)~ Dr. Dexter Rogers and from strain K-12 by l\ziss bI:iili Hrinsoo by a method previously descrihrd (1) Highly purified prepmxtions of hovine mam mary gland Klucosc-li-phospll:tte drhq-drogenase were kindly provided I)!, Air. Gordon .Julian. A partially purified hcxokinase (Type IV) M BS geueror~sly supplictl by the Sigma Chemical Company, Ortho nit.rophenyl p-n-galactoside \r>ts purchased from Sigma Chemical Co. lactose (c.1’. grade) \~as treated with ion-exchange resins to remove trnres of inorganic ions and crystallized from :rq,wwus ulcohol. lIethy p-u-gnl:lrtositlr W:IS prepared 1)~ st:ind:ad methods. The pH stat rlsed was :I 1%Funct,ion:tl Rccortling Titrator purchased from the International Instrument Company, Cmyon, California. The polnrimeter used ww a photoelectric model m:tnuf:tctured hy t.he Rudolph I’olxrimetcr Co. :tnd made :tvnilnl)le through the rourtwy of I)r. Johrl Schrll,n:\n
The experiments whose data are summarized in Figs. 1 rind 2 were carried out, as follo\vs. Ewh incubation had a total volume of 1.00 ml. and ~OJItniued 2.65 X 1Om3mg. rnzymc. The suhstratr: oNl’G, was present in :I concentration of 10m3 M. The buffers were 0.05 df TrispTht:. 1ncuh:ttion was for 10 min. at 25”. The hydrolysis of oKI% was estimat,ed by adding to t,hc 1.00.ml. incubntion smnples 0.4 ml. of M ?ja&O:, and measuring the optical density at 420 *np (4). Optical density or nbsorb atice units per IO-min. assay relmrted in tal)les or figurrs can hc converted int,o mpM/min. I,,v using a conversion factor. Thlls 21.7 m~df/min. corrcspends to 1.0 optical density unit. Tris-ThG I)utfers containing substrate were prepared as follows. Thioylj-colic acid, 0.05 ‘11, was m:ltle up in oxygen-free w:tt,er. To 10-m]. s:rml)les were added 50 ~1. oM’(: in pyridine (60 mg./ml.); solid nnalytir:tl gratlc Tris was added t 0 :i pH ne31 that desired, and the final pH was adjusted wit,11 a pII-stat. The titration w-as donr in :I nitrogen atmosphere, and solutions lucre stored t~ndcr nit rogcn.
The method used to meas,Ire the esteut. of 1:~ tow hydrolysi:, was ii spect rophotomet ric* enzymic rst,im:ation of the glu~osr 1”odlwctl. The eriz,vmic rcagent~ \v:*s prep:trcd 1)~ adding to 18.0 ml. of 0.05 If ‘l’riswrwt at r t)utirr, pH 7.0, 0.2 ml, of M hJg(:ll! , 0.4 ml. highly purifictl glllcose~(i-phosphate tlrhydrogenxw (1.7 mg./ml.), 0.4 ml. ‘l’l’r (20 mg./ml.). 0.4 III]. hrxokimrse (Sigma ‘I’ypr IV, 10 mg./ml. J :m(l 0.2 ml. .4T1’ (200 mg./ml.). Aliquots of 0.98 ml. bvere t em1~cr:~t~~rc-cc~r~ilil~r:it cd 31. ‘15” iu the silica ruvet t,c lwfore ILSC. This wagcut rollltl IN: stored 31 - 10” :intl thawed repeatedly bkithorit iqprecGal)lr tlrteriorntion alt bough long standing c:tusctl iniwt ivation. Addition of lo--’ moles glrirosc (5 ~1. of 0.0 :II glucose) caused :I rapid inrre;rse ill optical density :rt 310 mp as mc:tsurcd iu the ISwkman I)17 qwt rophot~onicter equipped with “thrrmo-spacers ” to m:rint:Gn a 1empcr:tture of 25”. The tot al A:iJ,, for t.his :miount of glucose was O.WO opt~iral density unit in less thari 10 min. Sinyr >Ig++ \V:LS rrquitwl t)y the glucose tlet,cction syst cm, it ~viis newss:try to inrut)at.c 1)uffcred lactose wmplcs with the enzyme in small t utws, to stop the rraction 1)~ pl~~nging the tribe in boiling \v:tter for :I-5 min. , and, :tftrr cooling, to unalyzc appropriatr :diquots. Aliquot size was chosen so that) first -order kinetics obtained in the glucose tlrt,ection system. I,nct ose solrLt,ioris of v:wying ronccnt,ratiofi were prepnrctl by diluting a cwnrcritr:ttrd stock solution of 1:tctosc bvitli O.OY M ‘I’riswtcrtntr hllfler, pII i.0. To :t 0.2S1nl. sample of such a solution iu :I smxll t ulw was atldcd 5 ~1. of 0.5 dl ?;:tCl or KC1, :m(l thr tulw W:LSrqlGlibr:rted at 25”. To this was uddetl r:ipidly ant1 with good mixing 20 ~1. enzyme sollltiori (0.132 mg), After 2.0 miu. the incuhntion tube ~v:ts plrulgctl into boiling wat,er to st,op rr~zymc xction :LII(~ then I)rought, to 25’. This \V:LS done in a staritl:ndized bv:iv so as t 0 minimize scvrlnl variewrre :~nalyzed ties of esperimcrit:11 error. _4liyuots ~~~r~tro1~l~otomctricall~as indicutrd prcviourly.
I~:xpwimcnts to dcterminc~ the approximate concentration of Mg++ needed to ohtail1 maximum hydrolysis rates arc exemplitied 1)~ IJig. 1. In the next set of experiments, summurized ill l’ig. 2, the cwnwntrations of both ,I’a+ and Mg++ were vnricd. It) may tw noted that, R’Ig++ is far more effective at low pH values. Further, it can be seen that the optimal pEI in the presence of ndequatc amounts of both ions, Sa+ and TV@++,XLS
27:;
\
\ \ 0 \
NoMg++od,jed
-----------v-l--
3
2
0
4
5
- log I Mg++l
0.60.5--
/
0 /
/o-. 0,
\
/ a? z 0.4-a g 0.3--
/
0.
0 /
‘\
, / /
\
AC0 ++o++
\ 0, O*
4.
/ / P’
0.2-m
/ 0. I ~~ /’
xxx *
*
,‘. Y ,“-
6.0
kii
,o-
*O
-o-.-o-.
3’
** o+
5.6
,‘\
OkX
%’
/
,O’ /
040’ 5.2
/
x x
o6.4
*’
+;o
0
-o-.--
-A’
6.6
>’
--0 7.2
7.6
6.0
6.4
PH
ti.8, hut at ION vnlucs of either, the pI1 optimum n-as raised to about 7.1. These data cmfim and extend those of Ref. (0). 15sw~tially the same values \vere ohbained with trkncetntr buffers a.s UWT ohtainrd
with tris-ThG huffcrs. This indknt ed that ThC; was 11ot inducing a11 Aig++ efkrt 1)~ cornplcx formntiml 1101’Tv:is it inlpnrt~nut, during the short. iuculmtiou period, to t alw rneasurcs iu order t 0 protect --SII groups.
However, stock enzyme solutions were always diluted with tris-ThG which had been prepared, and stored, in nitrogen. Another set, of experiments was done to establish a relation between Mg++ and Mn++ effects in such systems. n-0 essential difference could be detected bet’ween the rat,es attained in t,he presence of iYa+ and Mg++ and those attained in the presence of Sa+ and Mn++. The dat’a in Table I show the concentraTABLE THE
IXHIBITIOX
I
OF oXPG BY EDTA
HYDROLYSIS
IncubaGons contained IO+ 111oNPG Tris-acet,ate pH 7.0 and 1OW M Xa+. added.
in 0.05 M No hIg++
Optical density
EDTA M
,035 ,071 .200 .200
10-a 10-d 10-s 10-G
TABLE
THF, EFFECT HPI~OLYSIS
II
RIWWTIOS OF EDTA &GALACTOSIDASE
INHIBITION BY Na+
iTa+
OF
Optical density
.lf
,122 ,092 ,035
10-1
10-z 10-3
Incubations contained 1OW M oNPG in 0.05 M Tris-acetate pH 7.0 containing lo-’ M EDTA. TABLE REMOVAL OF EDT-4 p-GALAC,POSIDASE Mg++
III INHIBITION BY h3g++
OF
Optical density .~______.
__
T--
10-Q 10-s 10-4 10-S 10-o Ko EDTB
tion of EDTA which will cause inhibition. In the next experiment,, Table II, it will he seen t’hat raising the xa+ concentration tends to reverse EDTA inhibition slight,ly. In Table III t’he dat,a clearly illust,rate t,hat Mg++ removes EDTA inhibition and is consonaut, with t)he evidence of Fig. 2. Since the amount of enzyme present n-as identical in each incubation of t,his series, it, is evident t~hnt Mg++ does more than activate slightly even when adequate Iia+ is present. Figure 3 shows t’hat the maximum \pelocity of hydrolysis in t,he presence of Mg++ and Sa+ was no great’er t’han in the presence of Na+ alone, alt’hough the value of K, was substjantially reduced. The value of Tin,:,, in the presence of K+ was 2 X 1OP moles/ml./ min.; in the presence of Sa+, -l.% X 10Py. The values of K, were as follows: K+, 3.:3 X lo-” 111; Na+, 10-3; Na+ + Mg++, 3.7 X 10-d.
0.460 0.458 0.424 0.090 0.075 0.450
Incubations contained 1OW M o??PG in 0.05 M Tris-se&ate pH 7.0 containing 1OF M Xa+ and lo-’ M EDTA.
OF
10~s OF
ox
THE
LACTOSE:
With lactose as a substrate, no Xr[g++ requirement was observed. In t,he presence of 0.02 J1 K+ or n-a+, the presence of 1 X lop4 AT EDTA had no effect. In 3.3 X lo-” ill EDT‘4 and no added univalent ions, t’he enzymic acbivit’y was greater than 10 % of Ohe maximum rate observed in the presence of 0.02 JI li[+. Figure -2 shows that in relatively high concemrations of lactose the presence of K+ allowed a greater velocity of hydrolysis than Na+. In dilute solmions t,he difference was less marked. Indeed, there was appreciable act’ivity (about’ 30 % of maximum) when no ions were added. Values obtained by extrapolation of the points plot’ted in Fig. 4 were as follows. In the presence of ii-a+: K, , 3 X 1OF 111; l;‘,n,, , 7.8 X lO-7 moles/mm. In the presence of K+: Ki,, , 7.7 X 10-3; I’,,, , 1.7 X low. The availability of a photoelectric spectropolarimeter encouraged us to study enzymic lactose hydrolysis by measuring the changes in rotat’ion. The rotation of 0.1 ~11 lactose at 25” was easily and reproducibly measured to 0.002” (X13 mp). When hydrolysis was effected with enzyme, only
p-GALACTOSIDASE
900 600 c
-3
-2
-I
6
I
2
3
4
5
6
7
6
9
IO
rx lo-3hA ISI FIG. 3. l/71 versus l/S plot of oiYP(: centration of each ion 10-S M.
18
hpdrol,vsis
rates in various
ionic environments
at, pH 7.0. Con-
t
8 6
0 FIG. 4. l/a versus trntion 10e2 31.
40
80
l/S plot of lactose
120 hydrolysis
160 cont,rasting
200 effect
of Nnf
240
"WlU
and of I<+. Ionic
concew
276
ItEITHEL
slow changes were observable. Apparently (a!,@)-lactose, (CY,/3)-glucose, and P-galactose all have rotations which are nearly identical. Further, at, pH 7 the P,CY mutarot)ation of galactose is slow. Thus, it, seemed advisable t,o abandon this simple physical method in studies with lact,ose, but it was used wit)h success in examining anot,her suhstrat’e. Had cr-gnlactose been liberated, rotst,ional change should have been observable. The results suggest that the fl-anomcr is released, but, further careful experimentation will be required t,o establish this.
Since &galact,osidase action differed so greatly in ionic requirements in the foregoing st,udies, ot,her substrates were examined. Methyl p-n-galactoside was chosen because it, exhibits a low rotaCon but would yield upon hydrolysis a product of much great,er rotat,ion. Initial rates of change of rotation were determined so that t,he rate would be relatively unaffert)ed by p,cr mut’arot,ation. Observations mere made at 365 rnp and 25”. As in the case of lact’ose, no effect of Mg++ was observed. In experimental mixtures containing 0.1 111 substrate in 5 x lop4 111 sodillm phosphat,e buffer, pH 6.8 and 0.15 mg. enzyme/ml., t,he rates observed were slightly greater than in the corresponding potassium phosphate buffer. In tris-acet,at,e buffer, where no inorganic ions were added, t)he rate of change of rotation was almost nil. When 0.1 ~11 subst.rate in 0.05 -11 tris-acetat,e, pH 7.0 was used, and ions added as the chloride at lo-’ ill concentration, t,he rates in the mixtures containing IX+ were slightly greater than those containing Sa+. DISCCSSION
The evidence reported in t,his paper indicates that, (a) magnesium ion is necessary as an adjuart, or roenzyme only in the rapid hydrolysis of oXPG, (b) it reduces t’he K, value, and (c) it, does not raise I’,,, in the presence of adequate ?;a+. It follows t’hat neit,her Mg++ nor Mn++ can be considered as an essemial part, of t#his enzyme system nor can they be considered act,ivators for all
AND
KIM
subst’rates. In no experiment were we able to demonstrate any difference bet,ween the a&on of NIg++ and that of ,&In++. Further experimentation will be required t,o discover whet her Mg++ can be excluded vigorously from the enzyme wit,hout, loss of activity. et al. have stated [(I I), p. As MaImstrom 13-l] : “another t’ype of artifact. becomes more common wit,h highly purified enzymes, namely, that due to t,he metal ions stabilizing the protein in dilute solution.” Since the use of oI%PG as substrate allows assays at very low enzyme concentrations, it. must) be determined whether t,he NIg++ effect is a function of the protein csoncentration for this sub&ate. Apparent,ly previous workers have not excluded all NIg++ (or Mn++) since the pH at which maximum hydrolysis rates have been observed is more often recorded as 7. Figure 2 shows clearly that, absence of n!Ig++ can be not,ed by a decided increase in t,he pH value at, maximal act,ivity. It is apparent that :I small amount, of Mg++ is more effective at low pH values than at high pH values. This would speak against competition by protons for complexing. l’rcsumahly this effect is related to the pK’ of some group on the enzyme. Ion-binding studies are planned in order to oht,ain more evidence. The observations made on univalent ion effcrt,s c~onfirm the claims of Cohn and Monad (7) and may be useful in explaining some contradictions. It is obvious that. the relative effwt,s of Ka+ and Ii+ depend on the concentrations of t,hese ions as well as hydrogen ion. Kot enough data have been collected to make evident the relation bctween substrate and ion requirement. In a recent#ly report,ed case (12), Kf was sho\vn to have a greater effect’ on the K, \.alue than on l’,,;,, . It is evident that’ t’his cannot, be the case for p-galactosidase when lactose is the substrate, but) it may be true for ot,hrr subst,ances. It is tempt,ing to postulate t,hat. K+ or Sa+ can be bound in such a way as to diminish hydrogen bonding, which may comribute to the st,abilit,y of t)hc CC zyme-substrate complex. Stated alternatively, some dissociable group at the “a(Ative site” may funct8ion more cfficient~ly in the presence of an ion contribut>ing less to reso-
wuw stabilit’y. This hypothesis is susceptiMc to experimental testing a11d may (wlitrihute sometjhing to an understancl& of unixdent iou effwts.
Proteins” (Benesch et al., eds.). pp. “Bi-41. Academic Press, Sew York, 1059. 4. 11~~~:~~~~~, J., J. fkckrid. 60, 381 (1950). <5 I~CBY, s. il. BNI) LARl>Y. H. iz., J. .1ta. C’hou. sot. 75, 890 (1953). 0. 7. 8. 0
1, Hu, 2%. $. I,., WOIXE, R. (;., ANU J., .li~zh. Biochcnr. Nioph?/s. 81, 2. WALLESFELS, Ii., ZARSITX, 11. I,., BESI)ER, H.. ANLI KWER, RI., 331, ‘I50 (1959). :<. \li.41,1,ENFEI,S, Ii., iTI “Symposium
&ITlIEL,
F.
500 (1950). LAI-LE, (:., Niochew. %. on Sulfur
in
COFIES-BAZIRE, (+., .4s1) iLIosol), J., f’orupl. w/id. 232, 1515 (1951). I)., asI, (;LASS, B., “The i\lrchIO. ~~~:LRc)Y,W. anism of Enz,xxw Action,” pp. “87-00. Johns Hopkins l’rws, lMtimow, 1954. 13. (i., ~NI) ROSENBERG, A., :I& 11. &1AI,MSTRihl, uunres in Enzyrrcol. 21, 153 (1959). 12. 'I‘AHOR, H., AND WYN(:ARUEN. I,., ,I. h'id. (‘hew. 234, 1812 i1050).