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Isocitrate lyase from Pseudomonas indigofera I. Preparation, amino acid composition and molecular weight
~
BIOCHIMICA ET ,BIOPHYSICA AC-TA
~BA 65138 t~'~iI~:l~A:~'E LYASE FROM PSEUDOMONAS [NDIGOFERA L V ~ E - P ; ~ T I O N , AMINO ACID COMPOSITION AND MOLECULAR WEIGH'[ ~M~
~H[~( ~ ~ t ~ ~ U S H I I O
~-~nt
a . ~ BRUCE: A. McFADDEI~*'*
of C~¢a~a~:~, Washing~ 5tat~ University, Pullman, Wash. ( U.S,A.)
An imptove.d method for the purification of isocitrate lyase (Ds-isocitrate gi3~,~a~:date4ya:~e, EC 4.L3.I) from Pseudomonas indigofera is described. The enz3mae -W~[mr~ti~m is homogeneous by sedimentation and diffusion criteria and virtually ~:~.c:~e~eo~m I~;- gel-electrophoretic analysis. "the.~-dimentation coefficient (s~o,w),diffusion coefficient (D,0,w), partial specific ,0oin~e, ,~ole~adat weight, extinction coefficient at a8o m#, absorbancy ratio at ~ ,'~ g~g~m/t, atnd turnover number of crystalline isocitrate lyase are respectively, ~4t~" ~o~'~ ~¢, 3.87' Io'-? cm~/sec, 0.730 , 2,22- :o ~, 1.71o cm~/g, 2.o3, and 7300 moles #_y~yiate formed per rain per mole enzyme. "D~e a~fino acid composition of the enzyme was determined by ion-exchange cht*m~;,t%-¢ai~lky..All of the common amino acids are present in the enzyme. The *mryme cotlt~in~ zl half-cysfine residues, 255 basic amino acid residues, ~97 aspartic pbt~ aspa.ragil~e residues, and 240 glmamic plus glutamine residues per mole.
A, de~crilJtion of the crystallLsation of isocitrate lyase (Ds-isocitrate glyoxylate]yz~i~e, EC 4.I.3.I ) from Pseudomonas imligcfera has been published recently by McFAo~t~ A.~t.*How£sL This has made it possible to study its physical and chemical ~:b~r~ :teri~ti:cs and mechanism of cat alysis. However, some modifications of the method of p~,:r.fic~t[on and crystallization afford better reproducibility and a much better yield, arid a:ee fl~erJore particularly useful for large-scale preparations of enzyme. This comm~Jnicati~m de,~.:r~bes the modifications as well as studies of the amino acid compositi ~ a.nd several phy.~co~chemica] properties of isocitrate lyase from P. {ndigofera. Abbre~'iati,m- TEM, o.o~ M 'I'ri~, coataining I mM EDTA and t mMfl-mercaptoethanol. ()rl Jeave of al~l~c~ from the Ce~Ttl~! l~e~e~,rch Laboratory, Ajiuomoto Co., the., KawaO~t:tea~,e of ~b~nec from tile lnst,tufe of Apph~d M~erob~ology,Umversity of Tokyo, Japan, * " T~:*eau~thor ~o whom inquiries should be addresued.
g~gd~fm, IJi~phys. Acts, 96 {t965) II4--122
ISOClTRATE LYAStL PAR~" I
~1~
MATERLa-LS ~ND ~I/;THODS
Ma/en'a/s ~L-tsocitrate was prepared from the [actoae ( a l l ~ f r ~ ~¥hieh ~ a | ~ u ~ t of California:Corporation for Biochemical Rese~h~ ~ i ~ g[~xy|~e ~xl~,~,te
C~dture ¢nd p~@ar~ion of caftan P. indigofera ~ which Was L~)tated at Hopkins M~fine Stat~m~ P~,~c Gc~*s~, CalifA was used in the present studio. This bact~:t~u w~s g..~'a s ~ e g ~ , in t.~ follos~.Jngmedia at 30°: first culture~/n 36 rn! '~$o~3% Dif~x~~ t e;xtra,:t o¢~~. ~ha~¢~• for 24 h; second culture, in I-~ I of a bae~l i~,~.~ q -.~ ~ ., ~al~ ~ a n ~ ~ ~ t ~ i l ~ ~.~,.~~,~ sodium bat~q'ate and o , t % l ~ f ~ )~,~t e~lraet ~ h ~atk~r~ f ~ ~4 h~ th~tal~ d ~t~n culture, in 40 1 of an identical ,~.¢fltum but '~,~th ~.~: % Dif~x~3x~asl ~t~a~:~. ~and ~ no-z4 h ~sSth aeration. The cells (~',ei:g~. ah~ut ~oo g~ ~ g ~ t,~'v~ted ~B~c~mt~na~.~s centrffugation, washed once xsSth o.o5 ~ Tfis ~ptt 74L a,~d st~,~d ~t .......;.~L A ~ , ~ t ~ of the cell suspe~asion in 300 ml of 0.05 N Tri~ {pH 7.7L c:imt~$~i~ 3 ¢ ~ ~ < ~ h ~x~e treated in a Raytheon to-kcycte~ ~mic ~/l|atg~r ~s~th tap~wa~6, <~g~;~.~t at: fan|| power for 6 rain and then ctmtrifuged ;at io5 4~o ~ g¢~¢ t h~ 13~¢ ~ ! ~ a ~ . ~ ~ (crude extract or Fraction E) was stored in a free~er~ Enz),m¢ assay After the enzyme had been pmincuhat~ ~ t h ~$~ of GSlt f~v |o ~:~ at room tempexat~L~ein a total Votarae of 2~ ml ~a~taining g ~ ~ o~ ~g~g ~p|~ 7-7). and 6 ~m~ole.~of M~.~t~,the e~&vmati~ ~e:a~hm was inRggted by ~ke ~d~it~ ~'~f o , -~ m | of 0.04 M sodium l~r~-h~:itrate, at 3 ~ under ~ir ~¢ ~,~de~r N~, ~ h e n 1he ~ & ~ extract was a.~zyed), The reaction ~ s stopla~ by th, ~(Mtikm ~ff too rot. ~f ~#% (w~v) trichloroacettc acid after ~rOmln~ arid LO mi <~the, rc~t~m :~;X~t~ wCc~:~ X ; ~ } for glyoxylate by the ~th'od ,of McbL~t~-og~g~O HO.Wt~o ( ~ t ~ s ~ ~:n~i~ ~ k ~ as the amount of en~x~mecata|~ang the forn~itm~ff ~ ~at~.~e of g ~ L a l ~ f¢,.~ i~o~trate in x rain at 30* under th~ ab~ve ~xmd~th~¢~ S ~ 6 ¢ a~:t~v~ty ~g ¢ : x ~ a ~ as enzyme amirs ~ r rag of peotdn~ Protei~ x ~ C~a~i~,~t : s i ~ : ~ r t ~ - ~ ; ~ i i ~ hy the general method of ~VARt~t~G A ~ C I ~ I ~ A ~ | ~ ~ . ~ : ~ ' ~ . ~ ~:~°ao yeast nucleic acid a~d cU~tgtline ~g~ci~r~ieiyase ~ee l ~ : ' t , ~ RESULI~
•
amio, .
i :
!
~
:":
5~,~::~tti!_ ~¢ the: addition of ~ N acetic add with stinSng; and o.x volume of fresh g ~ m i ~ Stti:f~e~iltt~on ~Zomg/ml, pH 4~5-5.o)was slowly added. The precipitate a'~ t ~ l i by eentri~ugati!m~ The pH of the supematant solution was adjusted to ; ~ 7 7 ~ ~y t~e additiot~ of x M:Tris (pH 8.5), and the soluticn (PS) was brought to 0~4~ ~i:nr~ti¢m by the addition of 0.89 volume of saturated (NH4)~SO~solution i~ijt~ted to pff 7:7 with cone, NH~OH). The precipitate wa~ removed by centrifuga~ ~nd |he Supernatant .solution brought to 0:53 saturation by the further addition fh~¢~,:~5voh~me of tL'e saturated alkaline' (NH~)zS04 solution. The precipitate was c ~ K ~ by c~ntrifugation, and dissolved in TEM. After the protein concentration ~ been adjusted to 5-7 mg]mi with TEM, the solution (LP) was dialyzed overnight :a~. be£ore~ arid them placed:on a z cm × 3z cm column containing ! 4 g of DEAEi~t|t~l~ ~57~t~ mesh},, which had been previously washed consecutively with x N ~aO]g, ~ate~, 0.,O5M Tr~ {pl:l 7.7):, and TEM. A gradient elution was accompfished b~ ~t~lagt~t~i~:~o-,~,l ~ k e r s as the mixing chamber and reservoir. They were mounted ~tt t[f*'.,:.~e |t~ve] ~nd~¢~nnected by tubing so as to mz,intain hydrostatic equilibrium. .¥~.~til of 0,05 M NaCI containing 0,05 M Tris (pH 7.7), ~ mM EDTA and zmM fl-
3t'
E
o
r~:~ IsJ
~,0
Fro¢tton
6o
ro
fJo
Number
ti~g5 ~, t~r~p~rat;ive.seale chromatography of isocitrate lyase (dialyzed Fractiort I P on D E A F ~,it~:Jo~e. I totem (:~3o rag) was elated by gradually increasing the~ m01ari~y of NaC| front o.o 3 to 0.3 Ma.~ d,z,~:r~bed in the text. I t ~ t l , A~s0 nan; D ~ L q , enzyme activity; O - - © , enzyme activity divided: |:~y ;4 ~ ma~ " " "
Biod.h¢,~t. Biophys. Acta, 96 {~965} 114-t~2
i$0~ITRA~i~
i~¥ANi;ipARl,!i:i..:
~:: : ;
:.
t~ 7
solution (pH 7.7-8)~ The precipitate ~w.s removed by filtration with c h ~ l o t h . The filtrate was allowed to stand in a large petri dish at ~° for 3-4 d~ys until o.55 ,~taration was reached (desired decrease of weight in g ~ o.x8 x initial vohm~e in t~flL Ti~e crystalline material was collected b y centrifi|~:tion and dissoh'ed in TI~M to result h~ solution C. Table I shows ar~ example t~om rt~ults of the purification prortxhtre..In ~:verat TABLE I eaa~aaarm.~ or cavsr,~t.ms~ Isoel~aAte ~ a s s Starting material: 85 g of P, i~digofera (wet), For d~,,'l'ipti
L F r a c t i o n t~
eS10
e~'t"
a. 34. 5. 6.
t ~t o 5~o 397 17~ 3LS
4,6
~t~
5~ 5-1 ~~.¢3 z9
~ i.~ 3~ ~
33
1~
Supern~tant SH Ppt. P Solution PS Solution LP Pooled Fractior~s F
7. C ~ t a l l i n e m a t e r i a t
(Solution C)
a~, 7
II(m
" D e t e r m i n e d u n d e r N s {~'e M~rI~eL~LS .~.'I~ M ~ H o ~ ) ,
expeximents~ the foltox~ng attditional rt~ults were obiai~ett. The enzyme it~ Fraction E was sometimes re~-onabty stable at 50*.for Io rain even with~mt attdiiitm ~f MgCt~, which protected the purifiM enz3nme from heat inaeti~ti~lL tt~)~,x~, the ~(kliti~t~ of MgCI~ is recmmneLuted as a reasamable phi:aurOra. Nmletim~q i[h~r N~X?il:~R;~t~at 0.45 saturation in the first fn~ctionation ~ith { N t t ~ f ) ~ had s~:~neactivity, wNle tl~e supernatant solution at 0.55 c~atu~tion had little activi W in all ca~es~ Wher~ nucleic acid had not been remov~t b y treatment ~ t h protautiue s~tfate, ~ o ~ 1 ~ achieved N the next step,as ~,own l~yi t l ~ o f the ~ t i e o f a l ~ t ~ a c y at ;~N~.~u and 26o m~. In the cr3~taltization ptoces-s, a considerable ;+m~mm ~f i~:~-luifle titnW matexial was formed on the surfat~, o f t,he .'~m~lutlon~whle& m ~ h t hax~e l.~m ~urfit~:~
~tAI uluavo~!
~plt)~ ~
........
,. . . .
. . . .
:
In ,oneeXperiment the irt¢iU~on o f Str~ptom)X:i~ t e e ; ~ i ~ t ~ s~N~:~tm~t S H resulted in litile; c 2 t m ~ in sin'.iN aeti,¢~ty .or ~ * b ~ ! ~ y :rafi~ ,ai eS0 ~ N ~ nV,~
~;:~
I. SHItO~ T, SHtlO, B. A. MCFADDEN
|:~ ¢*;~:et~t e~I~r~m~.mtsthe crystallization step, w;hich results in some surface de~tt~:,tutlo~ !'~aSbeen r~placed by a refractionation procedure in which precipitate has | ~ i ~ e~-~lSe,:~edl!Mt¢iradji~sti~g Fractions F {about t mg protein per ml) to o.47-o.53 ~.$uratiem with .saturated INH~)~SO~ (pH 7.7). This has increased the field from ~,~Cfiong F from about 65 to 8o%..The product, though not crystatl:ine, was essentially ~:,rr~er~e~m:~ ~'<'i ~',/o minor component) by gel-electrophoretic criteria and had a ~p~fi¢.a~Cfivi~V 5f 314 OcCasiOnally.speCific activities as hi h as 4z- have been ob:: ~:~2~I:q~[ t ~ y trapidl'~ d~¢t!nea:tb the::usua[v-alues upon storage at --20" in TEN i A~ yet t~ere: i~[ti0 sati~fact0r~( exNanati6 ~ for this.
M Tris (pIt 7. 7) containing •i~.~O~,~I GSIi ,w~re,:.carri~dout:witlia SNnco. ~i0det E ultracentrifuge eauiDDed with
i ;: : i¸¸'" ; i ' ~ i.(i ' :. ' : (; ~ ; " t~e large: feff~etive griidient from: the meniscus was due to distribution of the nonpr~AeJt~buffer components in the ~entrJfugM field 0~pplied. The s~,~,~,calculated from the sdflieren pattern was 9,49" IO-~3 sec and was essentially the same when the solvent ~~ritained o,i M T~is (pH 7,7), and o.oi M GSH plus 3 mM MgC[~,Solvent of the latter compoSitkin was used in the diffusion studies. T!ae p~,rtiai specific v0h~me (~) ~:ffthe enzyme was calculated from th:~ weight p~xce~t:~ges of tli e amino acid residues and their respective specitic volumesSA It was o.73: 'l)ifft~sion studieg were performed in a Spinco Model H electrophore~s-diffnsion
ISOCITRATE LYASE. PART I
I 19
e n z y m e was dissolved in the solvent described earlier and the solution was equilibrated with the solvent overnight through a dialysis m e m b r a n e before the analyses. The diffusion coefficient, D~o,,,., was calculated as 3.87. Io '-~ cm~sec. Using s.,,~, D~+,~ and the estimated e, the molecular weight of isoeitrate Iyase was calculated as 2,22
•
I O 5.
Gd dectrophor~;sis In early experiments the clectr~phrting nmdium is shown m Fig, 4. It u a s i m l ~ i h l c t~ ttet+W~llilt~2
Origin Fig..~, Acry~amide -gel elet~tr~phorcsis of i~o('itra ~e lya~-. The ~-I i¢'y;u~o~lum. E 4? Appa r~ t ~s t'~, was preptured by a m o d i f i c a t i o n o f t|lg nlt, lho~t of NAYMOXt~ AND WAN~) ?, a~.~ll i~O-t'~lt s|icil:rs
"A..'l't"
equilibrated overnight ~t 3o~eV with o,~ M Iris ~-~mtainillg ~:~:~ M G.";t't, Then ~ta~ gttl}iw x~41~ replaced with tYesh i)~lf!l~tr,lipid a oo4!%as[)Itatli{ln Of i,~witrate lya~c ~dd~! l~ ~he sl~(, ~|~gt4xtio~fr~m left to right oecurr(~[ ti~r .., h ia a field ~:ff3o0 V wixt~ t~l~|~-t~llater ~'~l~Ug~'~'the gel c~tapart~wnl,,.. 3\) i(tcntify protein t~n(t.~ naphthylan~int- black was n,~.d~, the relative a m o u n t s ~ff isocitrate lyase ~md the nmre slowly mi~"a~i~zg minor c~m~= ponent by densitometric meth~ds so large xxz~s the difference in dye nptake. It is estimated t h a t the m i n o r eoml~ment is 1 ~ than z ~-, ~f the naaman"~,ne. Indexed the minor component m a y hax'e been derived from isocitrate 13~.~ it~cl|\
Dry wc~ghl and ul#avioM amdysis A dry xs'eight determination was performed on an aliq~o~ ~,f cry.~talth~e enzyme solution, which had beela t~L~.~ed th~o~tgh a .,%phade,x G~5o ~ c o a ~ ~) o A u a m h~h~ distilled x~ter. The ~ampte was lyophili~.~M a a d fhen fl~'th(~r dried to c¢~nstaat ~xqght at approx, x m ~ in a v a c u u m ow?no Another aliqu~t r+f the s~qation ~~f crystalline enz3qne was employed h~ s t u d y tiae ultravi~lct ah~t'pti,~'a sial'llama. |nSl~,cti~a of the Sl:n.wtrum at p i t 7-7 rev~;ais a. i~,ak at 27~ ~ 7 9 rap, as slmw~a in Fig, 5- The absorbancy ratio at "-,Soto 2~:~ n~p wa.-~ ~,1~32: ~,~1, The ~ at ~ n \ . ~ i s ~7zo cme.'g in o,o5 M Tris ( p H 7~7~ at z5L
A m i ~ add ~ml'+silion For amino acid analy~\% e~,',~ue ~ h u i o n . which had t.~en d e ~ | ~ I~y' l~:~s~age througl~ a ~ p h a d t . x e~lumn into distilled ~ e r ~ ~ s u~,d ~.~ the ,~mple, Sami@,¢ c o n t ~ n i n g approx, o,7 m g in 6 N HCt ~ t e h)~in~ty~¢~l a~: ~,.~5~ in ~att, d evacu:dcd tubes for t2., :t4, and 48 1~. C3~teine a n d e)~tine wer~ d e t e ~ i n ¢ ~ i .a~ rystc~t a c k k am| methi~mine as methioai~e sulfoac aficr D, rft~rmic a r k ! ~xb.tatk,~a ~ff th~.~ protei~a f,)~"
~O
I. SHIIO, T. SHIIO, B. A. MCFADDE~T
2~0
~t~O
270
~'80
290
300
~10
Wave Len0th in m/~
F~g. 5- UIt~aviolet ,'~bsorptlon spectrum of isoeitrate lyase. Crystalline enzyme was in 0.o5 M 'rris (pH 7~7), and the palh length was I.O cm.
TABLI~ I I A,~I~O :,¢ID COMPOSITION OF ISOClTRA'rELYASE HMGcystine was determined as cystcic acid, ~md methionine as methionine s~flfone (see text). For ~he me~d~od used for tryptophan determination see text.
Amino acid /esidue
lle¢overy after hydrolysis (izmoles ) Ie h
Asp The" Set Gill Pro Gty Ai~ Val lieu Lt;u T,cr Phe Lys His Arg NHn Cv~ Met Try
e4 h
48 h
o,571 0,559 0,536 o,3~3 o.3~18
o~339 0,258 0.695 o.z27 o.5Io o.765 o.375 o,18o 0.374 o,231 0:93 0-302 O"119 °'236 o.462
0,245 0.694 0,233 0,498 0.755 o,438 0.24I 0,406 0.235 0,205 0.328 O'I35 °'~73 0.479
0.249 o.691 o,217 0.509 o.738 0.452 0.239 0.399 0.243 o.2o~ 0.33~ O"132 0"264 0.602
Extrapolated or maximum value (pmoles)
Amino aaid per zza ooo g of protein (moles)
Nearest in$egr~l number e,,famino acid residues per molecMe of protein
0.572 0.347 0.260 0,697 o.z33 o,51o o.771 0.452 o.241 0.406 0.243 0.205 o.331 O'135 °-273 0.458 o.o6I °"I24 0.062
197 I~198 89.7 240.3 8o. 4 t76 266 156 83 140 83. 7 70.7 114 46'6 94 .I 158 2t 4 z'8 ~ ~.4
197 i~o 90 240 8o x76 266 156 8.3 14o 84 7t tt4 47 94 i58 2t 43 :t
Biochim, Bio.~hys. Aaa, 96 (~965) tI4-I2~2
isocrrlt~¢~ z , X ' n ~ PARr i .
.
.
.
.
' L ;
Z~]
=.5 h at 6# folimged by acid h~lrolysis s. ,Mniao acid anal~es were ~arried m,t with a Technicon Auto ~alyzer~. Tryptophaii.¢ontent Was esthratted by the method of G~tawi.S A~¢ MOgUl::3" from the ultravio!et absorption spectrum of emc~zne~lution in .o,~ M NaOH, ]%e
of th~e polation "he yidd content of the enzyme,: i : : ~ , = The recovery of protein ma.~ on the basis of ma:~ of ~a~dxtoadds plus N'.H~ recovered was &$%. 'The recov,~,~,of N in amino acids attd NHs ~ s .Sa% of the N-content deterralned h~lependent[y by dl.~tlt;:lt and se&sl~d~ti~m, The latter ~o,~ content w"as IS.j .,,~. Whether th e enz3,'rae h;.:..~sa N-:<4.chm~mlXnlentwlfich t h ~ :~.~t contain amino acids remains to be seen.
I)I~U.c$10N
The purification and c~tatlizatio~ proc~.urc for ~s~'i~rat~ l y ~ d ~ : r i l ~ l tLv MCFADDENAND HOWES~~ lncnfit~t ~tl~n|y ill t ~ ~||OWi~l~Wa~+S,:addition of heat treatment and pn~tamine sul~te tl'-~tnlen~; omL~i~n of the C~i~(PO~l;-gd t~atment; use of enz~anc precipitated in a n m ~ ' e r r,hnge of (NFI,)~SO~¢ o n ~ t r a ~ in both salt fractionations; tee da~,ion l~rocedu~ darirg ~x~|~mrichromatography; ~nd the, concentration pn~cedur¢ in the trb~talliza~tion:ste~. ~ the ~ i l ~ show, a ~ yiead of the ac tivi~" was.0btained in each step, ~pecJa-~v ha the t~tl~ part of tl~ pr~x,th~re~ The small chang,e ha SPeCif~:activi W as a ~ u l t of ~he ¢~aiF~ation st~p su~n~ts that the t~ak fracti~ns fn~m coluran daromatography are a | n ~ t pure. l'he fiua| enz6m~e pre.txacation is ~'~ential[y hon~:~neo~s ( < t % irfinor ~xamp~ne~t) k~-~ on sedimentation, diffusio, and ekn'~tr~vetic a n a l ) ~ The re~zox~, of e a t ' m e unih~ :is ~1% ~Table t)o 'I%~% l h ~ l~.~t hav.e bt~m the p~tein in the SUl~r~atemt fix~mto5 4 ~ ×~g, C~ark'; P, i ~ ¢ e r a is ala escdleat soul '~ of i~citrate lyase, w t f ~ is n~t s~irpfi.~ng when it ~ ~¢~i~kh~t¢~lthat er)~l:~lliue end)lie is obtaiit~._~l after onI.v 14*fi~ld ~tift£~ai~)n..
reaction, has a tltrtlt~It nulbt~r ~f $ ~ ( i l , ~ of f n ~ per mln per n~le .e~t~e) at ~ " an4 pH !.6 ( ~ i f , l.i)~
t,fl~ipht~.q~hcile dc.~vcd
:i:) , ::t,
:~
::N:i~tefa!iy aeNiaoN!e.dgeab,:~ks:ar6 :a!~6 afi~":i6~Dr:i:,l~:M; fi~' ~ g ~Vatlab~le:M~s;:i~;::H*RR,CR,:t6peaormflielgNr~le6t6phOretie g:*~N~iS;,:::Liv:ito .conduct ¢he: ~-ana!ySes/ThlS :invc.sfigation Vas • i ~ : ~ :by ::R~t~h: ~r~t;!@,ogo39.:fron~ ithe Nation~a :Insfitutff of ~ali~ ~ ! ~ re~e,ti,~h Career Deveiop~ent ~Award (No; I-K3-AI%Z68)from ~fhe ~+~Ot~ ,'ffA:~gy and lnfecliou,~ Di:~a,Ses,U.$, Public Health Service.
,$ f ) ~ § + a . ~ ~ W. C ~ T ~ , Biod~era. Z,, Mo (tO4t) 384. iJ~ :~[!~,~ ~ ~ & ~ j~ ~. E ~ L | ~ , i11 E. J, CoI~N .~ND J, T. EDSALL, Protei,s, Amino Acid~ and ~ "~ "L ~ V M ~ m ~
~ : ~ N M~rt.~Vt^LL, Science, i16 (1952) t42.
• ~'~ f).,~:tL ~,V, ~im~, J , Biol, Chem., Z19 (~95(') 6 t t .
BIOCHIMICA ET ,BIOPHYSICA AC-TA
~BA 65138 t~'~iI~:l~A:~'E LYASE FROM PSEUDOMONAS [NDIGOFERA L V ~ E - P ; ~ T I O N , AMINO ACID COMPOSITION AND MOLECULAR WEIGH'[ ~M~
~H[~( ~ ~ t ~ ~ U S H I I O
~-~nt
a . ~ BRUCE: A. McFADDEI~*'*
of C~¢a~a~:~, Washing~ 5tat~ University, Pullman, Wash. ( U.S,A.)
An imptove.d method for the purification of isocitrate lyase (Ds-isocitrate gi3~,~a~:date4ya:~e, EC 4.L3.I) from Pseudomonas indigofera is described. The enz3mae -W~[mr~ti~m is homogeneous by sedimentation and diffusion criteria and virtually ~:~.c:~e~eo~m I~;- gel-electrophoretic analysis. "the.~-dimentation coefficient (s~o,w),diffusion coefficient (D,0,w), partial specific ,0oin~e, ,~ole~adat weight, extinction coefficient at a8o m#, absorbancy ratio at ~ ,'~ g~g~m/t, atnd turnover number of crystalline isocitrate lyase are respectively, ~4t~" ~o~'~ ~¢, 3.87' Io'-? cm~/sec, 0.730 , 2,22- :o ~, 1.71o cm~/g, 2.o3, and 7300 moles #_y~yiate formed per rain per mole enzyme. "D~e a~fino acid composition of the enzyme was determined by ion-exchange cht*m~;,t%-¢ai~lky..All of the common amino acids are present in the enzyme. The *mryme cotlt~in~ zl half-cysfine residues, 255 basic amino acid residues, ~97 aspartic pbt~ aspa.ragil~e residues, and 240 glmamic plus glutamine residues per mole.
A, de~crilJtion of the crystallLsation of isocitrate lyase (Ds-isocitrate glyoxylate]yz~i~e, EC 4.I.3.I ) from Pseudomonas imligcfera has been published recently by McFAo~t~ A.~t.*How£sL This has made it possible to study its physical and chemical ~:b~r~ :teri~ti:cs and mechanism of cat alysis. However, some modifications of the method of p~,:r.fic~t[on and crystallization afford better reproducibility and a much better yield, arid a:ee fl~erJore particularly useful for large-scale preparations of enzyme. This comm~Jnicati~m de,~.:r~bes the modifications as well as studies of the amino acid compositi ~ a.nd several phy.~co~chemica] properties of isocitrate lyase from P. {ndigofera. Abbre~'iati,m- TEM, o.o~ M 'I'ri~, coataining I mM EDTA and t mMfl-mercaptoethanol. ()rl Jeave of al~l~c~ from the Ce~Ttl~! l~e~e~,rch Laboratory, Ajiuomoto Co., the., KawaO~t:tea~,e of ~b~nec from tile lnst,tufe of Apph~d M~erob~ology,Umversity of Tokyo, Japan, * " T~:*eau~thor ~o whom inquiries should be addresued.
g~gd~fm, IJi~phys. Acts, 96 {t965) II4--122
ISOClTRATE LYAStL PAR~" I
~1~
MATERLa-LS ~ND ~I/;THODS
Ma/en'a/s ~L-tsocitrate was prepared from the [actoae ( a l l ~ f r ~ ~¥hieh ~ a | ~ u ~ t of California:Corporation for Biochemical Rese~h~ ~ i ~ g[~xy|~e ~xl~,~,te
C~dture ¢nd p~@ar~ion of caftan P. indigofera ~ which Was L~)tated at Hopkins M~fine Stat~m~ P~,~c Gc~*s~, CalifA was used in the present studio. This bact~:t~u w~s g..~'a s ~ e g ~ , in t.~ follos~.Jngmedia at 30°: first culture~/n 36 rn! '~$o~3% Dif~x~~ t e;xtra,:t o¢~~. ~ha~¢~• for 24 h; second culture, in I-~ I of a bae~l i~,~.~ q -.~ ~ ., ~al~ ~ a n ~ ~ ~ t ~ i l ~ ~.~,.~~,~ sodium bat~q'ate and o , t % l ~ f ~ )~,~t e~lraet ~ h ~atk~r~ f ~ ~4 h~ th~tal~ d ~t~n culture, in 40 1 of an identical ,~.¢fltum but '~,~th ~.~: % Dif~x~3x~asl ~t~a~:~. ~and ~ no-z4 h ~sSth aeration. The cells (~',ei:g~. ah~ut ~oo g~ ~ g ~ t,~'v~ted ~B~c~mt~na~.~s centrffugation, washed once xsSth o.o5 ~ Tfis ~ptt 74L a,~d st~,~d ~t .......;.~L A ~ , ~ t ~ of the cell suspe~asion in 300 ml of 0.05 N Tri~ {pH 7.7L c:imt~$~i~ 3 ¢ ~ ~ < ~ h ~x~e treated in a Raytheon to-kcycte~ ~mic ~/l|atg~r ~s~th tap~wa~6, <~g~;~.~t at: fan|| power for 6 rain and then ctmtrifuged ;at io5 4~o ~ g¢~¢ t h~ 13~¢ ~ ! ~ a ~ . ~ ~ (crude extract or Fraction E) was stored in a free~er~ Enz),m¢ assay After the enzyme had been pmincuhat~ ~ t h ~$~ of GSlt f~v |o ~:~ at room tempexat~L~ein a total Votarae of 2~ ml ~a~taining g ~ ~ o~ ~g~g ~p|~ 7-7). and 6 ~m~ole.~of M~.~t~,the e~&vmati~ ~e:a~hm was inRggted by ~ke ~d~it~ ~'~f o , -~ m | of 0.04 M sodium l~r~-h~:itrate, at 3 ~ under ~ir ~¢ ~,~de~r N~, ~ h e n 1he ~ & ~ extract was a.~zyed), The reaction ~ s stopla~ by th, ~(Mtikm ~ff too rot. ~f ~#% (w~v) trichloroacettc acid after ~rOmln~ arid LO mi <~the, rc~t~m :~;X~t~ wCc~:~ X ; ~ } for glyoxylate by the ~th'od ,of McbL~t~-og~g~O HO.Wt~o ( ~ t ~ s ~ ~:n~i~ ~ k ~ as the amount of en~x~mecata|~ang the forn~itm~ff ~ ~at~.~e of g ~ L a l ~ f¢,.~ i~o~trate in x rain at 30* under th~ ab~ve ~xmd~th~¢~ S ~ 6 ¢ a~:t~v~ty ~g ¢ : x ~ a ~ as enzyme amirs ~ r rag of peotdn~ Protei~ x ~ C~a~i~,~t : s i ~ : ~ r t ~ - ~ ; ~ i i ~ hy the general method of ~VARt~t~G A ~ C I ~ I ~ A ~ | ~ ~ . ~ : ~ ' ~ . ~ ~:~°ao yeast nucleic acid a~d cU~tgtline ~g~ci~r~ieiyase ~ee l ~ : ' t , ~ RESULI~
•
amio, .
i :
!
~
:":
5~,~::~tti!_ ~¢ the: addition of ~ N acetic add with stinSng; and o.x volume of fresh g ~ m i ~ Stti:f~e~iltt~on ~Zomg/ml, pH 4~5-5.o)was slowly added. The precipitate a'~ t ~ l i by eentri~ugati!m~ The pH of the supematant solution was adjusted to ; ~ 7 7 ~ ~y t~e additiot~ of x M:Tris (pH 8.5), and the soluticn (PS) was brought to 0~4~ ~i:nr~ti¢m by the addition of 0.89 volume of saturated (NH4)~SO~solution i~ijt~ted to pff 7:7 with cone, NH~OH). The precipitate wa~ removed by centrifuga~ ~nd |he Supernatant .solution brought to 0:53 saturation by the further addition fh~¢~,:~5voh~me of tL'e saturated alkaline' (NH~)zS04 solution. The precipitate was c ~ K ~ by c~ntrifugation, and dissolved in TEM. After the protein concentration ~ been adjusted to 5-7 mg]mi with TEM, the solution (LP) was dialyzed overnight :a~. be£ore~ arid them placed:on a z cm × 3z cm column containing ! 4 g of DEAEi~t|t~l~ ~57~t~ mesh},, which had been previously washed consecutively with x N ~aO]g, ~ate~, 0.,O5M Tr~ {pl:l 7.7):, and TEM. A gradient elution was accompfished b~ ~t~lagt~t~i~:~o-,~,l ~ k e r s as the mixing chamber and reservoir. They were mounted ~tt t[f*'.,:.~e |t~ve] ~nd~¢~nnected by tubing so as to mz,intain hydrostatic equilibrium. .¥~.~til of 0,05 M NaCI containing 0,05 M Tris (pH 7.7), ~ mM EDTA and zmM fl-
3t'
E
o
r~:~ IsJ
~,0
Fro¢tton
6o
ro
fJo
Number
ti~g5 ~, t~r~p~rat;ive.seale chromatography of isocitrate lyase (dialyzed Fractiort I P on D E A F ~,it~:Jo~e. I totem (:~3o rag) was elated by gradually increasing the~ m01ari~y of NaC| front o.o 3 to 0.3 Ma.~ d,z,~:r~bed in the text. I t ~ t l , A~s0 nan; D ~ L q , enzyme activity; O - - © , enzyme activity divided: |:~y ;4 ~ ma~ " " "
Biod.h¢,~t. Biophys. Acta, 96 {~965} 114-t~2
i$0~ITRA~i~
i~¥ANi;ipARl,!i:i..:
~:: : ;
:.
t~ 7
solution (pH 7.7-8)~ The precipitate ~w.s removed by filtration with c h ~ l o t h . The filtrate was allowed to stand in a large petri dish at ~° for 3-4 d~ys until o.55 ,~taration was reached (desired decrease of weight in g ~ o.x8 x initial vohm~e in t~flL Ti~e crystalline material was collected b y centrifi|~:tion and dissoh'ed in TI~M to result h~ solution C. Table I shows ar~ example t~om rt~ults of the purification prortxhtre..In ~:verat TABLE I eaa~aaarm.~ or cavsr,~t.ms~ Isoel~aAte ~ a s s Starting material: 85 g of P, i~digofera (wet), For d~,,'l'ipti
L F r a c t i o n t~
eS10
e~'t"
a. 34. 5. 6.
t ~t o 5~o 397 17~ 3LS
4,6
~t~
5~ 5-1 ~~.¢3 z9
~ i.~ 3~ ~
33
1~
Supern~tant SH Ppt. P Solution PS Solution LP Pooled Fractior~s F
7. C ~ t a l l i n e m a t e r i a t
(Solution C)
a~, 7
II(m
" D e t e r m i n e d u n d e r N s {~'e M~rI~eL~LS .~.'I~ M ~ H o ~ ) ,
expeximents~ the foltox~ng attditional rt~ults were obiai~ett. The enzyme it~ Fraction E was sometimes re~-onabty stable at 50*.for Io rain even with~mt attdiiitm ~f MgCt~, which protected the purifiM enz3nme from heat inaeti~ti~lL tt~)~,x~, the ~(kliti~t~ of MgCI~ is recmmneLuted as a reasamable phi:aurOra. Nmletim~q i[h~r N~X?il:~R;~t~at 0.45 saturation in the first fn~ctionation ~ith { N t t ~ f ) ~ had s~:~neactivity, wNle tl~e supernatant solution at 0.55 c~atu~tion had little activi W in all ca~es~ Wher~ nucleic acid had not been remov~t b y treatment ~ t h protautiue s~tfate, ~ o ~ 1 ~ achieved N the next step,as ~,own l~yi t l ~ o f the ~ t i e o f a l ~ t ~ a c y at ;~N~.~u and 26o m~. In the cr3~taltization ptoces-s, a considerable ;+m~mm ~f i~:~-luifle titnW matexial was formed on the surfat~, o f t,he .'~m~lutlon~whle& m ~ h t hax~e l.~m ~urfit~:~
~tAI uluavo~!
~plt)~ ~
........
,. . . .
. . . .
:
In ,oneeXperiment the irt¢iU~on o f Str~ptom)X:i~ t e e ; ~ i ~ t ~ s~N~:~tm~t S H resulted in litile; c 2 t m ~ in sin'.iN aeti,¢~ty .or ~ * b ~ ! ~ y :rafi~ ,ai eS0 ~ N ~ nV,~
~;:~
I. SHItO~ T, SHtlO, B. A. MCFADDEN
|:~ ¢*;~:et~t e~I~r~m~.mtsthe crystallization step, w;hich results in some surface de~tt~:,tutlo~ !'~aSbeen r~placed by a refractionation procedure in which precipitate has | ~ i ~ e~-~lSe,:~edl!Mt¢iradji~sti~g Fractions F {about t mg protein per ml) to o.47-o.53 ~.$uratiem with .saturated INH~)~SO~ (pH 7.7). This has increased the field from ~,~Cfiong F from about 65 to 8o%..The product, though not crystatl:ine, was essentially ~:,rr~er~e~m:~ ~'<'i ~',/o minor component) by gel-electrophoretic criteria and had a ~p~fi¢.a~Cfivi~V 5f 314 OcCasiOnally.speCific activities as hi h as 4z- have been ob:: ~:~2~I:q~[ t ~ y trapidl'~ d~¢t!nea:tb the::usua[v-alues upon storage at --20" in TEN i A~ yet t~ere: i~[ti0 sati~fact0r~( exNanati6 ~ for this.
M Tris (pIt 7. 7) containing •i~.~O~,~I GSIi ,w~re,:.carri~dout:witlia SNnco. ~i0det E ultracentrifuge eauiDDed with
i ;: : i¸¸'" ; i ' ~ i.(i ' :. ' : (; ~ ; " t~e large: feff~etive griidient from: the meniscus was due to distribution of the nonpr~AeJt~buffer components in the ~entrJfugM field 0~pplied. The s~,~,~,calculated from the sdflieren pattern was 9,49" IO-~3 sec and was essentially the same when the solvent ~~ritained o,i M T~is (pH 7,7), and o.oi M GSH plus 3 mM MgC[~,Solvent of the latter compoSitkin was used in the diffusion studies. T!ae p~,rtiai specific v0h~me (~) ~:ffthe enzyme was calculated from th:~ weight p~xce~t:~ges of tli e amino acid residues and their respective specitic volumesSA It was o.73: 'l)ifft~sion studieg were performed in a Spinco Model H electrophore~s-diffnsion
ISOCITRATE LYASE. PART I
I 19
e n z y m e was dissolved in the solvent described earlier and the solution was equilibrated with the solvent overnight through a dialysis m e m b r a n e before the analyses. The diffusion coefficient, D~o,,,., was calculated as 3.87. Io '-~ cm~sec. Using s.,,~, D~+,~ and the estimated e, the molecular weight of isoeitrate Iyase was calculated as 2,22
•
I O 5.
Gd dectrophor~;sis In early experiments the clectr~ph
Origin Fig..~, Acry~amide -gel elet~tr~phorcsis of i~o('itra ~e lya~-. The ~-I i¢'y;u~o~lum. E 4? Appa r~ t ~s t'~, was preptured by a m o d i f i c a t i o n o f t|lg nlt, lho~t of NAYMOXt~ AND WAN~) ?, a~.~ll i~O-t'~lt s|icil:rs
"A..'l't"
equilibrated overnight ~t 3o~eV with o,~ M Iris ~-~mtainillg ~:~:~ M G.";t't, Then ~ta~ gttl}iw x~41~ replaced with tYesh i)~lf!l~tr,lipid a oo4!%as[)Itatli{ln Of i,~witrate lya~c ~dd~! l~ ~he sl~(, ~|~gt4xtio~fr~m left to right oecurr(~[ ti~r .., h ia a field ~:ff3o0 V wixt~ t~l~|~-t~llater ~'~l~Ug~'~'the gel c~tapart~wnl,,.. 3\) i(tcntify protein t~n(t.~ naphthylan~int- black was n,~.d~, the relative a m o u n t s ~ff isocitrate lyase ~md the nmre slowly mi~"a~i~zg minor c~m~= ponent by densitometric meth~ds so large xxz~s the difference in dye nptake. It is estimated t h a t the m i n o r eoml~ment is 1 ~ than z ~-, ~f the naaman"~,ne. Indexed the minor component m a y hax'e been derived from isocitrate 13~.~ it~cl|\
Dry wc~ghl and ul#avioM amdysis A dry xs'eight determination was performed on an aliq~o~ ~,f cry.~talth~e enzyme solution, which had beela t~L~.~ed th~o~tgh a .,%phade,x G~5o ~ c o a ~ ~) o A u a m h~h~ distilled x~ter. The ~ampte was lyophili~.~M a a d fhen fl~'th(~r dried to c¢~nstaat ~xqght at approx, x m ~ in a v a c u u m ow?no Another aliqu~t r+f the s~qation ~~f crystalline enz3qne was employed h~ s t u d y tiae ultravi~lct ah~t'pti,~'a sial'llama. |nSl~,cti~a of the Sl:n.wtrum at p i t 7-7 rev~;ais a. i~,ak at 27~ ~ 7 9 rap, as slmw~a in Fig, 5- The absorbancy ratio at "-,Soto 2~:~ n~p wa.-~ ~,1~32: ~,~1, The ~ at ~ n \ . ~ i s ~7zo cme.'g in o,o5 M Tris ( p H 7~7~ at z5L
A m i ~ add ~ml'+silion For amino acid analy~\% e~,',~ue ~ h u i o n . which had t.~en d e ~ | ~ I~y' l~:~s~age througl~ a ~ p h a d t . x e~lumn into distilled ~ e r ~ ~ s u~,d ~.~ the ,~mple, Sami@,¢ c o n t ~ n i n g approx, o,7 m g in 6 N HCt ~ t e h)~in~ty~¢~l a~: ~,.~5~ in ~att, d evacu:dcd tubes for t2., :t4, and 48 1~. C3~teine a n d e)~tine wer~ d e t e ~ i n ¢ ~ i .a~ rystc~t a c k k am| methi~mine as methioai~e sulfoac aficr D, rft~rmic a r k ! ~xb.tatk,~a ~ff th~.~ protei~a f,)~"
~O
I. SHIIO, T. SHIIO, B. A. MCFADDE~T
2~0
~t~O
270
~'80
290
300
~10
Wave Len0th in m/~
F~g. 5- UIt~aviolet ,'~bsorptlon spectrum of isoeitrate lyase. Crystalline enzyme was in 0.o5 M 'rris (pH 7~7), and the palh length was I.O cm.
TABLI~ I I A,~I~O :,¢ID COMPOSITION OF ISOClTRA'rELYASE HMGcystine was determined as cystcic acid, ~md methionine as methionine s~flfone (see text). For ~he me~d~od used for tryptophan determination see text.
Amino acid /esidue
lle¢overy after hydrolysis (izmoles ) Ie h
Asp The" Set Gill Pro Gty Ai~ Val lieu Lt;u T,cr Phe Lys His Arg NHn Cv~ Met Try
e4 h
48 h
o,571 0,559 0,536 o,3~3 o.3~18
o~339 0,258 0.695 o.z27 o.5Io o.765 o.375 o,18o 0.374 o,231 0:93 0-302 O"119 °'236 o.462
0,245 0.694 0,233 0,498 0.755 o,438 0.24I 0,406 0.235 0,205 0.328 O'I35 °'~73 0.479
0.249 o.691 o,217 0.509 o.738 0.452 0.239 0.399 0.243 o.2o~ 0.33~ O"132 0"264 0.602
Extrapolated or maximum value (pmoles)
Amino aaid per zza ooo g of protein (moles)
Nearest in$egr~l number e,,famino acid residues per molecMe of protein
0.572 0.347 0.260 0,697 o.z33 o,51o o.771 0.452 o.241 0.406 0.243 0.205 o.331 O'135 °-273 0.458 o.o6I °"I24 0.062
197 I~198 89.7 240.3 8o. 4 t76 266 156 83 140 83. 7 70.7 114 46'6 94 .I 158 2t 4 z'8 ~ ~.4
197 i~o 90 240 8o x76 266 156 8.3 14o 84 7t tt4 47 94 i58 2t 43 :t
Biochim, Bio.~hys. Aaa, 96 (~965) tI4-I2~2
isocrrlt~¢~ z , X ' n ~ PARr i .
.
.
.
.
' L ;
Z~]
=.5 h at 6# folimged by acid h~lrolysis s. ,Mniao acid anal~es were ~arried m,t with a Technicon Auto ~alyzer~. Tryptophaii.¢ontent Was esthratted by the method of G~tawi.S A~¢ MOgUl::3" from the ultravio!et absorption spectrum of emc~zne~lution in .o,~ M NaOH, ]%e
of th~e polation "he yidd content of the enzyme,: i : : ~ , = The recovery of protein ma.~ on the basis of ma:~ of ~a~dxtoadds plus N'.H~ recovered was &$%. 'The recov,~,~,of N in amino acids attd NHs ~ s .Sa% of the N-content deterralned h~lependent[y by dl.~tlt;:lt and se&sl~d~ti~m, The latter ~o,~ content w"as IS.j .,,~. Whether th e enz3,'rae h;.:..~sa N-:<4.chm~mlXnlentwlfich t h ~ :~.~t contain amino acids remains to be seen.
I)I~U.c$10N
The purification and c~tatlizatio~ proc~.urc for ~s~'i~rat~ l y ~ d ~ : r i l ~ l tLv MCFADDENAND HOWES~~ lncnfit~t ~tl~n|y ill t ~ ~||OWi~l~Wa~+S,:addition of heat treatment and pn~tamine sul~te tl'-~tnlen~; omL~i~n of the C~i~(PO~l;-gd t~atment; use of enz~anc precipitated in a n m ~ ' e r r,hnge of (NFI,)~SO~¢ o n ~ t r a ~ in both salt fractionations; tee da~,ion l~rocedu~ darirg ~x~|~mrichromatography; ~nd the, concentration pn~cedur¢ in the trb~talliza~tion:ste~. ~ the ~ i l ~ show, a ~ yiead of the ac tivi~" was.0btained in each step, ~pecJa-~v ha the t~tl~ part of tl~ pr~x,th~re~ The small chang,e ha SPeCif~:activi W as a ~ u l t of ~he ¢~aiF~ation st~p su~n~ts that the t~ak fracti~ns fn~m coluran daromatography are a | n ~ t pure. l'he fiua| enz6m~e pre.txacation is ~'~ential[y hon~:~neo~s ( < t % irfinor ~xamp~ne~t) k~-~ on sedimentation, diffusio, and ekn'~tr~vetic a n a l ) ~ The re~zox~, of e a t ' m e unih~ :is ~1% ~Table t)o 'I%~% l h ~ l~.~t hav.e bt~m the p~tein in the SUl~r~atemt fix~mto5 4 ~ ×~g, C~ark'; P, i ~ ¢ e r a is ala escdleat soul '~ of i~citrate lyase, w t f ~ is n~t s~irpfi.~ng when it ~ ~¢~i~kh~t¢~lthat er)~l:~lliue end)lie is obtaiit~._~l after onI.v 14*fi~ld ~tift£~ai~)n..
reaction, has a tltrtlt~It nulbt~r ~f $ ~ ( i l , ~ of f n ~ per mln per n~le .e~t~e) at ~ " an4 pH !.6 ( ~ i f , l.i)~
t,fl~ipht~.q~hcile dc.~vcd
:i:) , ::t,
:~
::N:i~tefa!iy aeNiaoN!e.dgeab,:~ks:ar6 :a!~6 afi~":i6~Dr:i:,l~:M; fi~' ~ g ~Vatlab~le:M~s;:i~;::H*RR,CR,:t6peaormflielgNr~le6t6phOretie g:*~N~iS;,:::Liv:ito .conduct ¢he: ~-ana!ySes/ThlS :invc.sfigation Vas • i ~ : ~ :by ::R~t~h: ~r~t;!@,ogo39.:fron~ ithe Nation~a :Insfitutff of ~ali~ ~ ! ~ re~e,ti,~h Career Deveiop~ent ~Award (No; I-K3-AI%Z68)from ~fhe ~+~Ot~ ,'ffA:~gy and lnfecliou,~ Di:~a,Ses,U.$, Public Health Service.
,$ f ) ~ § + a . ~ ~ W. C ~ T ~ , Biod~era. Z,, Mo (tO4t) 384. iJ~ :~[!~,~ ~ ~ & ~ j~ ~. E ~ L | ~ , i11 E. J, CoI~N .~ND J, T. EDSALL, Protei,s, Amino Acid~ and ~ "~ "L ~ V M ~ m ~
~ : ~ N M~rt.~Vt^LL, Science, i16 (1952) t42.
• ~'~ f).,~:tL ~,V, ~im~, J , Biol, Chem., Z19 (~95(') 6 t t .