dimer. For the ionic strength dependency studies, sodium chloride in concentrations of 0.008â0.25 M (Ultrex,. J.T. Baker, Inc.) was added to the solutions. For the.
P ROTE INS: St r u ct u r e, F u n ct ion , a n d Gen et ics 29:103–112 (1997)
Co m p u ta tio n a l, P u ls e -Ra d io ly tic , a n d S tru c tu ra l In v e s tig a tio n s o f Ly s in e -136 a n d Its Ro le in th e Ele c tro s ta tic Tria d o f Hu m a n Cu ,Zn S u p e ro x id e D is m u ta s e Cin d y L. F is h e r,1 D ia n e E. Ca be lli,2 Ro be rt A. Ha lle w e ll,3 P a u l B e ro za ,1 Te re n c e P. Lo ,1 Eliza be th D . Ge tzo ff,1 a n d J o h n A. Ta in e r 1 * 1 Departm en t of M olecu lar B iology, T h e S cripps R esearch In stitu te, L a J olla, Californ ia 2 Departm en t of Ch em istry, B rook h aven N ation al L aboratory, L on g Islan d , N ew York 3 Departm en t of B ioch em istry, Im perial College, L on d on S W7 2AZ , Un ited Kin gd om
AB S T R AC T Ke y c h a rg e d re s i d u e s i n Cu ,Zn s u p e ro x id e d is m u ta s e (Cu ,Zn S OD ) p ro m o te e le c tro s ta tic s te e rin g o f th e s u p e ro x id e s u bs tra te to th e a c tiv e s ite Cu io n , re s u ltin g in d is m u ta tio n o f s u p e ro x id e to o x y g e n a n d h y d ro g e n p e ro x id e . Ly s -136, a lo n g w ith th e a d ja c e n t re s id u e s Glu -132 a n d Glu -133, fo rm s a p ro p o s e d e le c tro s ta tic tria d c o n tribu tin g to s u bs tra te re c o g n itio n . Hu m a n Cu ,Zn S OD s w ith s in g le -s ite re p la c e m e n ts o f Ly s -136 by Arg , Ala , Gln , o r Glu o r w ith a trip le -s ite s u bs titu tio n (Glu -132 a n d Glu -133 to Gln a n d Ly s -136 to Ala ) w e re m a d e to te s t h y p o th e s e s re g a rd in g c o n tribu tio n s o f th e s e re s id u e s to Cu ,Zn S OD a c tiv ity. Th e s tru c tu ra l e ffe c ts o f th e s e m u ta tio n s w e re m o d e le d c o m p u ta tio n a lly a n d v a lid a te d by th e X-ra y c ry s ta llo g ra p h ic s tru c tu re d e te rm in a tio n o f Cu ,Zn S OD h a v in g th e Ly s -136-to Glu re p la c e m e n t. B ro w n ia n d y n a m ic s s im u la tio n s a n d m u ltip le -s ite titra tio n c a lc u la tio n s p re d ic te d m u ta n t re a c tio n ra te s a s w e ll a s io n ic s tre n g th a n d p H e ffe c ts m e a s u re d by p u ls e -ra d io ly tic e x p e rim e n ts . Ly s -136-t o -Glu c h a rg e re v e rs a l d e c re a s e d d is m u ta tio n a c tiv ity 50%fro m 2.2 ! 10 9 to 1.2 ! 10 9 M !1 s "1 d u e to re p u ls io n o f n e g a tiv e ly c h a rg e d s u p e ro x id e , w h e re a s c h a rg e -n e u t ra li z i n g s u b s t i t u t i o n s (Ly s -136 to Gln o r Ala ) h a d a le s s d ra m a tic in fl u e n c e . In c o n tra s t, th e trip le -m u ta n t Cu ,Zn S OD (a ll th re e c h a rg e s in th e e le c tro s ta tic tria d n e u tra lize d ) s u rp ris in g ly d o u ble d th e re a c tio n ra te c o m p a re d w ith w ild -ty p e e n zy m e bu t in tro d u c e d p h o s p h a te in h ibitio n . Co m p u ta tio n a l a n d e x p e rim e n ta l re a c tio n ra te s d e c re a s e d w ith in c re a s in g io n ic s tre n g th in a ll o f th e Ly s -136 m u ta n ts , w ith c h a rg e re v e rs a l h a v in g a m o re p ro n o u n c e d e ffe c t th a n c h a rg e n e u t ra li z a t i o n , i m p ly i n g t h a t lo c a l e le c t ro s ta tic e ffe c ts s till g o v e rn th e d is m u ta tio n ra te s . Mu ltip le -s ite titra tio n a n a ly s is s h o w e d th a t d e p ro to n a tio n e v e n ts th ro u g h o u t th e e n zy m e a re lik e ly re s p o n s ible fo r th e g ra d u a l d e c re a s e in S OD a c tiv ity a bo v e p H 9.5 a n d p re d ic te d a ! 1997 WILE Y-LISS, INC.
p K a v a lu e o f 11.7 fo r Ly s -136. Ov e ra ll, Ly s -136 a n d Glu -132 m a k e c o m p a ra ble c o n tribu tio n s to s u bs tra te re c o g n itio n bu t a re le s s c ritic a l t o e n zy m e fu n c tio n th a n Arg -143, w h ic h is both m e chanistically and e le ctrostatically e ssential. Thus, the se que nce -conse rve d re sidue s of this e le ctrostatic triad are e vide ntly im portant solely for their electrostatic properties, w hich maintain the high catalytic rate and turnove r of Cu,Zn SOD w hile simultaneously providing specificity by selecting against binding by othe r anions. P roteins 29:103–112, 1997.
! 1997 Wile y-Liss, In c. Ke y w o rd s : B ro w n ia n d y n a m ic s ; m o le c u la r re c o g n i t i o n ; s i t e -d i re c t e d m u t a g e n e s is ; fa c ilita te d d iffu s io n ; c ry s ta l s tru c tu re IN TROD U CTION Cu ,Zn su per oxide dism u t a se (SOD) is a n en zym e cr it ica l t o t h e a n t ioxida n t pr ocesses in cells. It is fou n d pr edom in a n t ly in eu ca r yot ic cells a n d pr ot ect s t h em fr om su per oxide r a dica ls for m ed a s a bypr odu ct of a er obic m et a bolism .1 Th is en zym e is of gr ea t biologica l in t er est du e t o it s lin ka ge t o gen et ic disea ses, su ch a s fa m ilia l a m yot r oph ic la t er a l scler osis (FALS)2,3 a n d Down ’s syn dr om e,4 a n d it s likely in volvem en t in a n t ia gin g m ech a n ism s.5,6
Gr a n t spon sor : NIH ; Gr a n t n u m ber s: GM-39345, GM-37684, a n d GM-23658; Gr a n t spon sor : NSF ; Gr a n t n u m ber : DIR882385; Gr a n t spon sor : NIH F ellowsh ips; Con t r a ct gr a n t n u m ber s: GM-11612 a n d H L-07695; Gr a n t spon sor s: Medica l Resea r ch Cou n cil of Ca n a da , Ch ir on Cor por a t ion , a n d Wellcom e Tr u st . Cu r r en t a ddr ess: Cin dy L. F ish er, St r u ct u r a l Bioin for m a t ics, In c., 10929 Tech n ology P la ce, Sa n Diego, CA 92127. *Cor r espon den ce t o: J oh n A. Ta in er, Th e Scr ipps Resea r ch In st it u t e, Depa r t m en t of Molecu la r Biology, 10550 Nor t h Torr ey P in es Roa d, La J olla , CA 92037. Received 1 Novem ber 1996; Accept ed 8 Apr il 1997
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Cu ,Zn SOD, a h om odim er wit h 153 a m in o a cid r esidu es (in t h e h u m a n en zym e),* a n d on e copper a n d on e zin c ion per su bu n it , a ct s by ca t a lyzin g t h e dism u t a t ion of O ·2 ! t o m olecu la r oxygen a n d h ydr ogen per oxide. Th e r ea ct ion is diffu sion lim it ed, wit h a r a t e con st a n t on t h e or der of 2 " 10 9 M !1 s !1 ,7,8 a n d is fa cilit a t ed by elect r ost a t ic for ces.9–15 Com pu t a t ion a l elect r ost a t ic st u dies of bovin e Cu ,Zn SOD u sin g a sim ple Cou lom bic r ela t ion sh ip 13 a n d ot h er m et h ods 11,14,15 su ggest ed t h a t t h e elect r ost a t ic t r ia d of Glu -132 (r esidu e 130 in bovin e Cu ,Zn SOD), Glu -133 (bovin e r esidu e 131), a n d Lys-136 (bovin e r esidu e 134), a s well a s Ar g-143 (bovin e r esidu e 141), a r e im por t a n t in dir ect in g t h e lon gr a n ge a ppr oa ch of su per oxide t o t h e en zym e’s a ct ive sit e.13 E xper im en t a l eviden ce a lso im plica t es a ll of t h ese r esidu es a s bein g in volved in elect r ost a t ic gu ida n ce (r efs. 16–20 a n d r efer en ces t h er ein ). P r eviou sly, we exper im en t a lly a n d com pu t a t ion a lly con fir m ed t h e r a t e-en h a n cin g effect s of m u t a t in g Glu 133 in h u m a n Cu ,Zn SOD t o eit h er a n eu t r a l or posit ively ch a r ged r esidu e,19 a s pr edict ed by a n a lysis of t h e bovin e X-r a y cr yst a llogr a ph ic st r u ct u r e. 13 P oisson -Bolt zm a n n elect r ost a t ic ca lcu la t ion s su ggest ed t h a t t h e in cr ea se in r a t e a r ose fr om su bst a n t ia l a lt er a t ion s in t h e elect r ost a t ic pot en t ia l a r ou n d t h e en zym e in t h e m u t a t ed pr ot ein s.19 Repla cem en t of Ar g-143 in t h e h u m a n en zym e wit h a n y ot h er a m in o a cid r esidu e pr oved t o be det r im en t a l in a ll ca ses t est ed.20 We n ow r epor t t h e r esu lt s of sim ila r a n a lyses of t h e m u t a t ion of Lys-136 in h u m a n Cu ,Zn SOD t o a r gin in e (K136R), a la n in e (K136A), glu t a m in e (K136Q), or glu t a m ic a cid (K136E ). We a lso exa m in e t h e r ole of t h e Glu -132-Glu -133-Lys-136 t r ia d by a n a lyzin g t h e t r iple m u t a n t E 132Q-E 133QK136A. METHOD S S ite -D ire c te d Mu ta n ts Th e wild-t ype en zym e wa s m a de in a yea st expr ession syst em a n d t h e sin gle-m u t a n t s K136R, K136A, K136Q, K136E , a n d t r iple-m u t a n t E 132Q-E 133QK136A wer e m a de in a per ipla sm ic E sch erich ia coli expr ession syst em a s descr ibed pr eviou sly.20 All wer e ba sed on t h e syn t h et ic h u m a n SOD gen e t h a t in corpor a t es t h e Cys-6-t o-Ala a n d Cys-111-t o-Ser (C6A/ C111S) t h er m ost a ble m u t a t ion s.21,22 Th is m u t a n t en zym e m a in t a in s t h e r a t e con st a n t -vs.-pH pr ofile a n d a ct ive sit e st r u ct u r e of wild-t ype h u m a n SOD.23 Th e in t egr it y of ea ch m u t a t ion wa s ver ified by sequ en cin g t h e en codin g DNA. Spect r oph ot om et r ic a n d dr y weigh t m ea su r em en t s wer e u sed t o det erm in e pr ot ein con cen t r a t ion s.
*Th r ou gh ou t t h is pa per, we will be u sin g t h e n u m ber in g of h u m a n Cu ,Zn SOD.
Ex p e rim e n ta l Ra te Me a s u re m e n ts Th e su per oxide dism u t a t ion r a t es of wild-t ype h u m a n SOD a n d it s Lys-136 m u t a n t s wer e m ea su r ed in pu lse-r a diolyt ic exper im en t s ca r r ied ou t wit h t h e 2-MeV va n der Gr a a ff gen er a t or a t Br ookh a ven Na t ion a l La bor a t or ies a s descr ibed pr eviou sly.24 Su per oxide r a dica ls wer e gen er a t ed in a ir /oxygen -sa t u r a t ed a qu eou s solu t ion s 25 con t a in in g 10 m M sodiu m for m a t e (Sigm a Ch em ica l Co.) a n d wa t er t h a t wa s pu r ified by dist illa t ion followed by filt r a t ion t h r ou gh a Millipor e u lt r a pu r ifica t ion syst em . An y a dven t it iou s m et a l ion s pr esen t in t h e solu t ion t h a t m igh t dism u t e su per oxide a n d skew r esu lt s wer e sca ven ged by t h e a ddit ion of 100 µM et h ylen edia m in et et r a a cet ic a cid (E DTA, Sigm a ). Sodiu m dih ydr ogen ph osph a t e (10 or 50 m M, Ult r ex, J .T. Ba ker, In c.) wa s a dded a s a bu ffer in g a gen t in t h e sin gle-m u t a n t st u dies. F or t h e E 132Q-E 133QK136A m u t a n t st u dy, H E P E S wa s u sed a s a bu fferin g a gen t t o a void ph osph a t e in h ibit ion .19 At om ic a bsor pt ion spect r oscopy wa s u sed t o det erm in e t h e level of a ct ivit y-essen t ia l copper pr esen t a n d t h u s t h e con cen t r a t ion of a ct ive en zym e. Deca y of su per oxide wa s m on it or ed spect r oph ot om et r ica lly a t 245–270 n m in t h e pr esen ce of 1–10 µM O ·2 ! a n d 1–10 µM SOD. Beca u se SOD is dim er ic in vivo, ca t a lyt ic r a t e con st a n t s wer e ca lcu la t ed fr om t h e a ct ive en zym e con cen t r a t ion s det er m in ed by h a lvin g t h e copper ion con cen t r a t ion s m ea su r ed wit h a t om ic a bsor pt ion spect r oscopy (P ye-Un ica m or GBC in st r u m en t ). Th is a dju st m en t gives t h e r a t e con st a n t per dim er. F or t h e ion ic st r en gt h depen den cy st u dies, sodiu m ch lor ide in con cen t r a t ion s of 0.008–0.25 M (Ult r ex, J .T. Ba ker, In c.) wa s a dded t o t h e solu t ion s. F or t h e pH depen den cy st u dies, t h e pH wa s a dju st ed by eit h er Na OH (99.999% pu r e, GF S Ch em ica ls) or H 2 SO 4 (Ar ist a r, BDH Ch em ica l Co.). Th e en zym es wer e n ot dea ct iva t ed over t h e pH r a n ges u sed, beca u se m ea su r em en t s on ea ch SOD solu t ion bega n a t a ppr oxim a t ely pH 5, wer e con t in u ed t o on e pH ext r em e, r ever sed t o t h e ot h er ext r em e, a n d t h en br ou gh t ba ck t o a ppr oxim a t ely pH 5 a ga in wit h n o sign ifica n t loss in a ct ivit y. Ele c tro s ta tic Ca lc u la tio n s a n d B ro w n ia n D y n a m ic s S im u la tio n s H ea vy (n on h ydr ogen ) a t om posit ion s wer e t a ken fr om on e of t h e five dim er s in t h e a sym m et r ic u n it of t h e t h r ee-dim en sion a l X-r a y cr yst a llogr a ph ic st r u ct u r e of t h e h u m a n (C6A/C111S) SOD. 23 P ola r (n it r ogen a n d oxygen ) a t om s wer e pr ot on a t ed wit h t h e pr ogr a m In sigh t II (Molecu la r Sim u la t ion s, In c., Sa n Diego, CA). St r u ct u r es of t h e m u t a n t s K136A, K136Q, a n d K136E wer e gen er a t ed by r epla cin g t h e lysin e wit h t h e a ppr opr ia t e r esidu e wit h in In sigh t II a n d en er gy-m in im izin g t h e r esu lt in g coor din a t es wit h Discover (Molecu la r Sim u la t ion s, In c.) on a Con vex
LYS-136 OF SUP E ROXIDE DISMUTASE
C240 m in isu per com pu t er a s pr eviou sly descr ibed.20 Th e side ch a in of K136E wa s n eu t r a lized t o a void sever e dist or t ion s of t h e st r u ct u r e du e t o st r on g ch a r ge r epu lsion s by glu t a m ic a cids 132 a n d 133. Th e r esu lt a n t st r u ct u r es a r e a ll st er eoch em ica lly r ea son a ble a n d a ssu m e n o gr oss r ea r r a n gem en t s (m ovem en t s of m or e t h a n 1 Å or so in h ea vy a t om posit ion s) u pon m u t a t ion (see F ig. 1). E lect r ost a t ic pot en t ia ls a r ou n d t h e fou r pr ot ein s wer e ca lcu la t ed wit h ver sion 2.1.1 of t h e Un iver sit y of H ou st on Br own ia n Dyn a m ics (UH BD) pr ogr a m developed by J .D. Ma du r a a n d M.E . Da vis in colla bor a t ion wit h J .A. McCa m m on ,26 r u n n in g on a Con vex C240 a n d t h e Cr a y Y-MP 4E a t Scr ipps. Th e pr ogr a m det er m in es t h e elect r ost a t ic for ces fr om t h e lin ea rized P oisson -Bolt zm a n n equ a t ion a t poin t s on a gr id a r ou n d t h e pr ot ein . P a r a m et er s descr ibed pr eviou sly wer e u sed in t h is wor k a s well.20 Wit h in t h e pr ot ein , a dielect r ic con st a n t (#) of 2 wa s u sed, wh er ea s a n # of 78 wa s a ssu m ed ext er n a l t o t h e pr ot ein , a n d dielect r ic bou n da r y sm oot h in g wa s a pplied. P a r t ia l ch a r ges fr om AMBE R,27 a ssu m in g a pH of 7, wer e u sed for t h e pr ot ein a t om s, wit h $2 ch a r ges on bot h t h e copper a n d zin c ion s, a n d a n over a ll ch a r ge of !1 on H is-63, wh ich liga t es bot h t h e copper a n d zin c. H ist idin e r esidu es wer e a ssu m ed t o be n eu t r a l, except for H is-43, wh ich is pr edict ed t o be posit ively ch a r ged fr om h ydr ogen -bon din g pa t t er n s. A 128 " 128 " 128 gr id wit h 1.0-Å spa cin g wa s con st r u ct ed, cen t er ed a r ou n d t h e SOD dim er for t h e P oisson Bolt zm a n n ca lcu la t ion . E lect r ost a t ic for ces ou t side t h e gr id bou n da r ies wer e com pu t ed ba sed on a Debye-H u¨ ckel r epr esen t a t ion of SOD a s a 30-År a diu s sph er e wit h n et ch a r ge cor r espon din g t o t h e ch a r ge of t h e dim er (!4 for wild-t ype, !6 for ch a r gen eu t r a lizin g m u t a n t s K136A a n d K136Q, a n d !8 for ch a r ge-r ever sin g m u t a n t K136E ). E lect r ost a t ic pot en t ia l va lu es wer e ca lcu la t ed a t t h e gr id poin t s (see F ig. 3) for t h e wild-t ype a n d ea ch m u t a n t a t fou r differ en t ion ic st r en gt h s: 0.025, 0.075, 0.150, a n d 0.260 M. Br own ia n dyn a m ics sim u la t ion s wer e r u n wit h pa r a m et er s a ssign ed a s descr ibed by Sin es et a l.11 a n d ou r pr eviou s wor k.19,20 Th e h ydr odyn a m ic r a dii of 2.05 Å a n d 28.5 Å wer e a ssign ed t o O ·2 ! a n d SOD, r espect ively. Tr a ject or ies wer e st a r t ed a t r a n dom poin t s on a 41.5-Å sph er e cen t er ed on t h e dim er a n d con t in u ed u n t il r ea ct ion cr it er ia wer e m et or u n t il exit fr om t h e syst em pa st a 500-Å sph er e wa s fou n d. Rea ct ion wa s defin ed a s a ppr oa ch of t h e O ·2 ! wit h in 7.0 Å of eit h er copper sit e of t h e dim er. Collision s bet ween t h e pr ot ein a n d O ·2 ! wer e det ect ed wit h a n exclu sion gr id ca lcu la t ed by in cr ea sin g t h e va n der Wa a ls r a dii of t h e pr ot ein a t om s by 1.5 Å t o in clu de t h e r a diu s of a su per oxide oxygen a t om . Tim est eps wer e in cr ea sed fr om 0.10 ps wit h in 40 Å of t h e dim er cen t er t o 0.25 ps bet ween 40 a n d 100 Å, a n d 1.0 ps ou t side of 100 Å.
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p K a D e te rm in a tio n s fo r Mu ltip le Titra tio n S ite s To in vest iga t e t h e r ela t ion sh ip bet ween t h e a ct ivit y of SOD a n d it s ch a r ge dist r ibu t ion , we ca lcu la t ed t h e t it r a t ion cu r ve of t h e pr ot ein by u sin g con t in u u m elect r ost a t ic t h eor y. Th is m odel for pr ot ein t it r a t ion is ba sed on t h e a ssu m pt ion t h a t t h e ch a n ge in a n a m in o a cid’s pK a wh en it is bou n d t o a pr ot ein is pr in cipa lly elect r ost a t ic in or igin , a n d t h a t t h e en er gy sh ift s of t h e pr ot on a t ed a n d u n pr ot on a t ed for m s of t h e r esidu e fr om t h eir va lu es in solu t ion ca n be ca lcu la t ed fr om a con t in u u m elect r ost a t ic m odel.28–32 Th e ca lcu la t ion s wer e ba sed on t h e X-r a y cr yst a llogr a ph ic coor din a t es,23 wit h m odifica t ion s descr ibed in t h e pr eviou s sect ion . At om ic pa r t ia l ch a r ges wer e t a ken fr om Discover,33 a n d va n der Wa a ls r a dii wer e t a ken fr om t h e PARSE pa r a m et er set . 34 Region s in a ccessible t o a sph er ica l pr obe of 1.4-Å r a diu s wer e a ssign ed a n # of 4, wh er ea s a ccessible r egion s wer e a ssign ed a n # of 80. Th e fr ee en er gies of t h e differ en t pr ot on a t ion st a t es wer e det er m in ed fr om a Gr een ’s fu n ct ion for m a lism t h a t is ba sed on a fin it e-differen ce solu t ion t o t h e lin ea r P oisson -Bolt zm a n n equ a t ion (see Ber oza a n d F r edkin 35 for fu r t h er det a ils). In pr in ciple, t h e a ver a ge pr ot on a t ion of a sit e is det er m in ed fr om a Bolt zm a n n a ver a ge over t h e 2 N pr ot on a t ion st a t es of t h e pr ot ein , wh er e N is t h e n u m ber of t it r a t in g sit es. In pr a ct ice, t h is is fea sible on ly for sm a ll syst em s, a n d a ppr oxim a t ion m et h ods m u st be u sed wh en t h e n u m ber of sit es exceeds a ppr oxim a t ely 20. A Mon t e Ca r lo m et h od 29 wa s u sed t o sa m ple t h e st a t ist ica lly m ost im por t a n t st a t es in t h e dist r ibu t ion , a n d t h e a ver a ge pr ot on a t ion of ea ch r esidu e fr om t h is sa m plin g wa s det er m in ed. Th is pr ocedu r e wa s con du ct ed a t pH in cr em en t s of 0.2, yieldin g a t it r a t ion cu r ve for ea ch r esidu e fr om wh ich t h e n et ch a r ge on t h e pr ot ein wa s obt a in ed. Cry s ta llo g ra p h ic S tru c tu re D e te rm in a tio n o f K136E Mu ta n t Cr yst a ls of t h e K136E m u t a n t of h u m a n SOD wer e gr own by t h e h a n gin g dr op va por diffu sion m et h od u n der con dit ion s sim ila r t o t h ose u sed t o obt a in cr yst a ls of t h e C6A/C111S pr ot ein .36 K136E SOD cr yst a ls a r e isom or ph ou s t o t h ese la t t er cr yst a ls, gr owin g in spa ce gr ou p C222 1 wit h u n it cell dim en sion s a % 205.2, b % 167.0, c % 145.5 Å a n d h a vin g 10 su bu n it s in t h e a sym m et r ic u n it 23 . 306,185 X-r a y diffr a ct ion da t a in t en sit ies wer e m ea su r ed a t bea m lin e 7-1 of t h e St a n for d Syn ch r ot r on Ra dia t ion La bor a t or y by u sin g ph osph or im a gin g pla t es (Ma r Resea r ch ). Th ese in t en sit ies wer e sca led a n d m er ged t o pr odu ce 58,307 u n iqu e r eflect ion s (99.6% com plet e t o 2.85-Å r esolu t ion ) wit h a m er gin g R -fa ct or of 10.5%. An in it ia l differ en ce elect r on den sit y m a p wa s obt a in ed by u sin g ph a ses ca lcu la t ed fr om t h e isom orph ou s 2.5-Å-r esolu t ion st r u ct u r e of C6A/C111S h u -
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m a n SOD.23 E xa m in a t ion of t h is elect r on den sit y m a p a llowed m odelin g of t h e posit ion s of t h e 10 m u t a t ed Glu -136 side ch a in s. Th e m odel wa s r efin ed a ga in st a ll diffr a ct ion da t a bet ween 8.0- a n d 2.85-Å r esolu t ion by u sin g X-P LOR, 37 wit h m a n u a l fit t in g of side ch a in s u sin g Xfit .38 E a ch of t h e 10 su bu n it s wa s r efin ed in depen den t ly wit h ou t a n y n on cr yst a llogr a ph ic sym m et r y r est r a in t s. Th e fin a l m odel h a s a n R -fa ct or of 18.9% a n d a fr ee R -fa ct or of 25.7% wit h a n over a ll RMS devia t ion fr om idea l geom et r y of 0.017 Å for bon d dist a n ces a n d 1.7° for bon d a n gles. RES U LTS Ac tiv e S ite Arc h ite c tu re a n d th e Ele c tro s ta tic Tria d Lys-136 lies in t h e m iddle of t h e ext en ded ‘‘elect r ost a t ic’’ loop (r esidu es 121–144) con n ect in g t h e t wo C-t er m in a l &-st r a n ds of Cu ,Zn SOD. Th is r esidu e sit s on t h e ou t er lip of t h e a ct ive sit e ch a n n el lea din g t o t h e ca t a lyt ic copper ion a n d is a bou t 11 Å a wa y fr om t h e copper. Lys-136 is h igh ly exposed t o solven t a n d for m s sa lt br idges t o t h e n eigh bor in g Glu -132 a n d Glu -133, cr ea t in g a n elect r ost a t ica lly im por t a n t t r ia d a bove t h e a ct ive sit e Cu ion . In t h e en er gy-m in im ized m odel st r u ct u r es of K136A, K136Q, a n d K136E , t h e side ch a in con for m a t ion s of t h e m u t a t ed r esidu es gen er a lly followed t h a t of wild-t ype Lys-136 (F ig. 1), a lt h ou gh , wit h ou t t h e ch a r ged in t er a ct ion wit h Glu -133, t h is r esidu e is n ot a s h igh ly con st r a in ed a n d is likely m or e flexible in a solven t en vir on m en t . (As seen in F ig. 2, a r a n ge of side-ch a in con for m a t ion s for Glu -136 wa s obser ved a m on g t h e 10 in depen den t su bu n it s in t h e X-r a y cr yst a llogr a ph ic st r u ct u r e of t h e K136E m u t a n t .) Th e m odeled side ch a in s did n ot h a ve u n fa vor a ble st er ic or elect r ost a t ic in t er a ct ion s a n d a ppea r ed su fficien t ly a ccu r a t e for t h e pu r poses of t h e ca lcu la t ion s descr ibed h er ein . E lect r on ic, cir cu la r dich r oic, a n d elect r on pa r a m a gn et ic r eson a n ce st u dies con fir m t h a t m u t a t ion of t h is r esidu e t o a n y of t h e t h r ee a m in o a cid r esidu es list ed a bove does n ot siza bly a lt er t h e a ct ive sit e st r u ct u r e.16 Th e det er m in a t ion of t h e cr yst a l st r u ct u r e of t h e K136E m u t a n t fu r t h er va lida t es t h is poin t . Visu a l com pa r ison of Glu -136 in t h e t wo m odels a n d in t h e 10 su bu n it s fr om t h e X-r a y cr yst a llogr a ph ic st r u ct u r e sh owed t h a t t h e con for m a t ion of t h e m odeled side ch a in lies wit h in t h e set of cr yst a llogr a ph ic con for m a t ion s (F ig. 2). As con fir m a t ion , ea ch K136E SOD su bu n it in t h e cr yst a l st r u ct u r e wa s com pa r ed a ga in st ea ch of t h e ot h er n in e su bu n it s. Aft er su per posit ion , t h e RMS devia t ion of a ll m a in -ch a in a t om s bet ween ea ch pa ir of su bu n it s com pa r ed r a n ged fr om 0.27 t o 0.57 Å. Th e RMS devia t ion of a ll m a in -ch a in a t om s bet ween t h e t wo su bu n it s of t h e m in im ized K136E SOD dim er wa s 0.28 Å. Su per posit ion of t h ese t wo m odel su bu n it s wit h ea ch of t h e 10 su bu n it s of t h e cr yst a l st r u ct u r e r esu lt ed in a n RMS devia t ion of a ll m a in -ch a in
Fig. 1. SOD subunit fold, active site metal ions, and relative positions of side chains at position 136 in wild-type X-ray crystallographic structure and models of mutants. The protein backbone is indicated by the green ribbon, with the electrostatic loop colored magenta and the disulfide loop colored gold. The wild-type Lys side chain is shown in white, that of the Ala mutant in yellow, that of the Gln mutant in blue and that of the Glu mutant in red. The copper and zinc ion positions are indicated by the orange and blue spheres, respectively. The side chains of the other two residues of the electrostatic triad, Glu-132 and Glu-133, and of the mechanistically important Arg-143 are also displayed in magenta. Figure was constructed by using the program Ribbons50 converted by A. Shah for rendering with AVS.51
a t om s r a n gin g fr om 0.26 t o 0.50 Å. Th u s, t h e a t om ic posit ion s of t h e m in im ized K136E SOD st r u ct u r a l m odels u sed for elect r ost a t ic a n a lyses fa ll well wit h in t h e r a n ge obser ved exper im en t a lly in t h e cr yst a l st r u ct u r e. E ven t h e elect r ost a t ic gr ids ca lcu la t ed for t h e K136E m u t a n t cr yst a l st r u ct u r e vs. t h e m odel, wh ose over a ll st r u ct u r e wa s der ived fr om t h e wildt ype cr yst a l st r u ct u r e, wer e n ot ver y differ en t . As sh own in F igu r e 4, differ en ces bet ween t h e m odel a n d cr yst a llogr a ph ic st r u ct u r e of t h e elect r ost a t ic pot en t ia l a r ou n d t h e glu t a m a t es a t 136 in bot h m on om er s wer e sm a ller t h a n t h ose seen fr om sidech a in sh ift s (a r isin g fr om va r ia t ion s in t h e sidech a in con for m a t ion s of flexible solven t -exposed r esidu es in t h e wild-t ype a n d m u t a n t cr yst a l st r u ct u r es) elsewh er e in t h e dim er. p H P ro fi le s o f En zy m e Ra te s P lot s of pH vs. r a t e for t h e wild-t ype en zym e a n d t h e m u t a n t s pr ovide a com pa r ison of r ela t ive a ct ivit ies a n d t h eir pH depen den cies a n d in dica t e t it r a t ion of cr it ica l r esidu es. Th e pH depen den ce of t h e en zym a t ic r a t e in wild-t ype SOD follows a pa t t er n of a fa ir ly con st a n t r a t e fr om pH 5–8, wit h a sh a r p dr op a bove a bou t pH 9.5 (see F igs. 5 a n d 6). In som e pr eviou s ca ses, sh ift s in pK a va lu es a n d n ew t it r a t ion even t s wer e som et im es seen in m u t a t ed en zym es. 19,20 Th e Lys-136 m u t a n t s a n d t h e t r iple-m u t a n t E 132QE 133Q-K136A, h owever, fa ll in t o t h e t ypica l pH
LYS-136 OF SUP E ROXIDE DISMUTASE
Fig. 2. Energy-minimized model structures of the K136E mutant subunits superimposed with the 10 subunits found in the asymmetric unit of the X-ray crystallographic structure of the K136E mutant. The two subunits from the model are indicated in yellow and cyan, whereas the pairs of subunits from each of the
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five dimers are indicated in magenta, green, orange, red, and blue. The position of the copper and zinc are indicated by orange and blue spheres, respectively. The overall orientation of the subunit in this figure matches that in Figure 1.
depen den ce pa t t er n seen for t h e wild-t ype en zym e a n d t h e m a jor it y of m u t a n t s. In t h e SODs wit h sin gle m u t a t ion s a t posit ion 136, t h e r ea ct ion r a t es decr ea sed wit h decr ea sin g posit ive ch a r ge a r ou n d t h e a ct ive sit e (F ig. 3), a s expect ed for loca l elect r ost a t ic a t t r a ct ion of t h e n ega t ively ch a r ged su bst r a t e: posit ively ch a r ged Lys ! Ar g ' n eu t r a l
Fig. 3. Slices through the electrostatic potential grids of (a) the wild-type SOD dimer and (b) the K136E mutant. The slices pass through the copper ion at the bottom of the active site channel of one subunit. Positive potential is indicated in blue, and negative in red. The color changes on the vertical plane show the decrease in attractive positive potential (blue) upon charge reversal of Lys136. C( traces of the SOD dimer backbones are indicated by white tubes; copper and zinc ion positions by orange and blue spheres, respectively. Figure was rendered with the program AVS,51 with tubes generated by the module MCS TUBES (written by Y. Chen and A. Olson).
Fig. 4. Differences in the electrostatic potential grids of the model and X-ray crystallographic structures of the K136E mutant. The C( traces of the two subunits are shown with the larger radius tubes. The position of the mutated side chains in each subunit is indicated by the smaller radius tubes in the upper left and bottom right. Areas of higher negative potential (or less positive potential) in the crystal structure are shown in red, and higher positive potential (or less negative potential) are shown in blue.
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Fig. 5. Reaction rate dependence on pH for wild-type and single-mutant enzymes. At pH 8, the wild-type enzyme (W) had the highest activity followed by the K136R mutant (M); mutants K136Q (U) and K136A (V) had intermediate rates, and K136E (Q) the lowest. Data were obtained in solutions of 0.05 M sodium dihydrogen phosphate and 0.01 M sodium formate, except in the wild-type in which 0.01 M phosphate was used. In the mutant enzyme solutions, 100 µM EDTA were added to chelate any free copper ions. Concentrations of SOD varied from 2.7 to 6.6 µM.
Ala ! Gln ' n ega t ively ch a r ged Glu (F ig. 5). Repla cem en t of t h e posit ively ch a r ged Lys wit h a n ega t ively ch a r ged Glu dr opped t h e a ct ivit y by a lit t le less t h a n h a lf, fr om a bou t 2.2 " 10 9 t o a bou t 1.2 " 10 9 M !1 s !1 a t ph ysiologica l pH , a m u ch sm a ller effect t h a n t h a t seen for ch a r ge-n eu t r a liza t ion a n d ch a r ge-r ever sa l m u t a t ion s of Ar g-143,20 bu t of r ou gh ly com pa r a ble m a gn it u de wit h t h a t seen for ch a r ge-n eu t r a liza t ion m u t a t ion s of Glu -132.19 Th e t r iple-m u t a t ion E 132QE 133Q-K136A ga ve t h e sa m e r esu lt s a s t h e sin gle m u t a t ion E 132Q, dou blin g t h e r a t e com pa r ed wit h wild-t ype, bu t a lm ost h a lvin g t h e r a t e com pa r ed wit h t h e dou ble m u t a t ion E 132Q-E 133Q (F ig. 6). In ot h er wor ds, n eu t r a lizin g Lys-136 (K136A) n ega t ed t h e in cr ea se in a ct ivit y ga in ed by n eu t r a lizin g Glu 133 (E 133Q), r esu lt in g in a n en zym e wit h t h e sa m e a ct ivit y a s t h e E 132Q m u t a n t . Io n ic S tre n g th D e p e n d e n c ie s o f En zy m e Ra te s Respon se of en zym e a ct ivit y t o ch a n ges in t h e ion ic st r en gt h of t h e pr ot ein ’s solven t en vir on m en t pr ovides a n in dica t ion of t h e r ole of elect r ost a t ic in t er a c-
Fig. 6. Reaction rate dependence on pH for triple mutant E132Q-E133Q-K136A and related mutant enzymes. At pH 8, the wild-type enzyme (W) had the lowest activity. Triple-mutant E132QE133Q-K136A (M) and single-mutant E132Q (Q) had similar activities. Double-mutant E132Q-E133Q (U) had the highest rate. Data were obtained in solutions of 0.5 mM HEPES and 0.01 M sodium formate. In the mutant enzyme solutions, 100 µM EDTA were added to chelate any free copper ions. Concentrations of SOD varied from 2.8 to 8.3 µM.
t ion s in det er m in in g t h e r a t e. In t h e ca se of h u m a n Cu ,Zn SOD, t h e over a ll wild-t ype pr ot ein in it s dim er ic for m h a s a n et for m a l ch a r ge of !4 a t pH 7, wh er ea s t h e su per oxide r a dica l h a s a ch a r ge of !1. If t h e en zym e’s a ct ion is in flu en ced m a in ly by globa l elect r ost a t ic effect s, t h e over a ll r epu lsive ch a r ge of t h e en zym e sh ou ld be scr een ed fr om t h e in com in g su per oxide a s t h e ion ic st r en gt h of t h e solu t ion is in cr ea sed. On t h e ot h er h a n d, if loca l elect r ost a t ic for ces dom in a t e, t h e elect r ost a t ic a t t r a ct ion of t h e posit ively ch a r ged a ct ive sit e wou ld be m a sked wit h in cr ea sin g ion ic st r en gt h , a n d t h e r ea ct ion r a t e wou ld decr ea se. E xper im en t a l eviden ce fr om t h e bovin e en zym e sh owed t h e la t t er effect ,9 wh ich wa s fou n d t o be t r u e for t h e h u m a n en zym e a s well.19,20 Th e ion ic st r en gt h depen den cies of t h e wild-t ype a n d m u t a t ed h u m a n SODs sh own in F igu r e 7 a r e det er m in ed by plot t in g t h e log of t h e obser ved r a t e con st a n t (k ) a s a fu n ct ion of t h e squ a r e r oot of ion ic st r en gt h (I 1/2 ). (Th e I 1/2 t er m is der ived fr om t h e Debye-H u¨ ckel lim it of t h e Br øn st ed-Bjer r u m equ a -
LYS-136 OF SUP E ROXIDE DISMUTASE
Fig. 7. Effect of ionic strength (I) on reaction rates as measured experimentally at pH 8 by pulse radiolysis (solid symbols) and computationally (open symbols) from Brownian dynamics simulations. Experimentally, the wild-type (W) enzyme, the most active, was most affected by ionic strength, the electrically neutral mutants K136A (M) and K136Q (U) less so, and the K136E (Q) mutant the least affected. Computationally, the ionic strength effects and relative activities of the wild-type (X) and mutant enzymes K136A (N), K136Q (V), and K136E (S) were very similar to those determined experimentally.
t ion .20 ) As obser ved in pr eviou sly st u died SOD m u t a n t s,19 t h e r ea ct ion r a t e decr ea sed wit h in cr ea sin g ion ic st r en gt h in a ll of t h e Lys-136 m u t a n t s, im plyin g t h a t loca l elect r ost a t ic effect s a lso gover n t h e su per oxide dism u t a t ion of t h ese m u t a t ed pr ot ein s. Neu t r a liza t ion of t h e posit ively ch a r ged Lys-136 decr ea sed t h e ext en t of t h is effect sligh t ly a n d ch a r ge r ever sa l even m or e. Th e degr ee t o wh ich t h ese m u t a t ion s a lt er ed ion ic st r en gt h depen den ce wa s a bou t t h e sa m e a s t h a t seen for m u t a t ion s of a ct ivesit e Ar g-143.20 Br own ia n dyn a m ics sim u la t ion s wit h t h e UH BD pr ogr a m sh owed t h e a bilit y t o pr edict r ea ct ion r a t es a n d t h e effect s of ion ic st r en gt h on t h e m u t a t ed en zym es. P r eviou s wor k by Sin es et a l.,11 wh ich qu it e a ccu r a t ely pr edict ed t h e a m ou n t of dr op in en zym e a ct ivit y on n eu t r a liza t ion a n d ch a r ge r ever sa l of Lys-136, a lso in dica t ed a decr ea se in t h e effect s of ion ic st r en gt h for r esidu e 136 (75% of wild-t ype u pon n eu t r a liza t ion , 35% u pon ch a r ge r ever sa l). Ou r ca lcu -
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Fig. 8. The overall calculated charge of wild-type SOD as a function of pH from multiple-site titration analysis. The shape of the curve at high pH is similar to the pH dependence of the activity (see Fig. 5), which suggests that the falloff in protein activity at high pH may result from the change in net protein charge as protons are dissociated.
la t ion s on r esidu e 136 ga ve sim ila r va lu es, wit h a n ion ic st r en gt h decr ea se of 75% a n d 62% of wild-t ype for K136A a n d K136Q, r espect ively, a n d 21% for K136E . Th ese wer e in good a gr eem en t wit h t h e exper im en t a l va lu es of 64 a n d 69% for K136A a n d K136Q, r espect ively, a n d 34% for K136E . As fou n d in pr eviou s wor k,11,19,20,39 t h e a bsolu t e r a t es wer e overest im a t ed in t h e ca lcu la t ion s; h owever, t h e Br own ia n dyn a m ics ca lcu la t ion s do give excellen t r ela t ive r a t es for m u t a n t s in wh ich elect r ost a t ics is t h e m a jor r ole pla yed by t h e m u t a t ed r esidu e a n d ca n t h u s be u sed t o est im a t e it s elect r ost a t ic con t r ibu t ion . p K a B e h a v io rs a n d Ele c tro s ta tic Co n tribu tio n s Th e ca lcu la t ed t it r a t ion cu r ve for t h e wild-t ype en zym e is sh own in F igu r e 8. Th e declin e in over a ll pr ot on a t ion in t h e pr ot ein m im ics t h e declin e in r a t e a bove pH 9.5 (com pa r e wit h wild-t ype cu r ve in F ig. 5). Th e sh a pes of t h e cu r ves a t pH va lu es gr ea t er t h a n a bou t 7.5 a r e sim ila r, a lt h ou gh t h e exper im en t a l r a t e fa lls off m or e r a pidly t h a n t h e ca lcu la t ed pr ot on a t ion st a t e a t h igh er pH . Below pH 7, t h e
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ca lcu la t ed t it r a t ion cu r ve sh ows a n in cr ea se in pr ot on a t ion a s t h e a spa r t ic a n d glu t a m ic a cid r esidu es a r e n eu t r a lized (F ig. 8). Ra t es a t low pH (F ig. 5) do n ot r eflect t h is pr ot on a t ion , su ggest in g t h a t t h e n ea r-opt im a l elect r ost a t ic a t t r a ct ion of su bst r a t e by t h e wild-t ype en zym e’s a ct ive sit e is n ot a ffect ed by pr ot on a t ion elsewh er e in t h e m olecu le. Th e m u lt iplesit e t it r a t ion a n a lysis a lso yields pK a va lu es for in dividu a l r esidu es ba sed on t h e elect r ost a t ic en vir on m en t . F or exa m ple, ger m a n e t o discu ssion s below, r esidu es Ar g-143, Lys-122, a n d Lys-136 wer e fou n d t o h a ve a ver a ge pK a va lu es of 10.7, 11.0, a n d 11.7, r espect ively. Wh en m u lt iple-sit e t it r a t ion a n a lysis wa s a pplied t o t h e R143K m u t a n t , t h e lysin e h a d a n a ver a ge ca lcu la t ed pK a of a bou t 6.9. D IS CU S S ION Sin gle m u t a t ion s a t Lys-136 in h u m a n Cu ,Zn SOD give en zym a t ic r a t e ch a n ges com pa r a ble wit h r a t e ch a n ges du e t o m u t a t ion s a t Glu -132 a n d Glu -133, su ppor t in g t h e h ypot h esis t h a t t h e elect r ost a t ic t r ia d of Glu -132, Glu -133, a n d Lys-136 pr ovides a n elect r ost a t ic n et wor k st eer in g t h e in com in g su per oxide r ea ct a n t in t o t h e a ct ive sit e. Ch a r ge-n eu t r a liza t ion a n d ch a r ge-r ever sa l m u t a n t s of t h ese r esidu es sh ow ion ic st r en gt h depen den cies a n d pH pr ofiles con sist en t wit h t h is h ypot h esis. Th e t r iple-m u t a n t E 132QE 133Q-K136A displa yed com pa r a ble beh a vior a n d sh owed t h a t t h e elect r ost a t ic effect s of t h e con st it u en t m u t a t ion s wer e a ddit ive. Th ese t h r ee r esidu es, wh ich a r e h igh ly sequ en ce con ser ved in kn own m a m m a lia n a n d ot h er eu ca r yot ic Cu ,Zn SODs,40 wor k in con cer t t o pr ovide a fa vor a ble elect r ost a t ic en vir on m en t t h a t con t r ibu t es t o t h e su bst r a t e r ecogn it ion a n d specificit y of t h e en zym e. Th e pr esen ce of Glu 133 pr even t s t h e bin din g of la r ge a n ion s, su ch a s ph osph a t e, in t h e a ct ive sit e.19 Lys-136 cou n t er a ct s Glu -133’s n ega t ive ch a r ge, t h er eby im pr ovin g t h e lon g-r a n ge a t t r a ct ion for a n ion ic species. Glu -132 h elps focu s a n ion ic su bst r a t es t owa r d t h e a ct ive sit e by pr ovidin g a r epu lsive n ega t ive ch a r ge ou t side t h e a ct ive sit e ch a n n el. Som e deba t e h a s a r isen a s t o wh ich specific r esidu e or r esidu es a ct u a lly con t r ibu t e t o t h e declin e in a ct ivit y obser ved in m ost SODs a t a r ou n d pH 9.5.16,17 Lysin es a r e kn own t o be in volved in t h e a ct ivit y a n d ion ic st r en gt h depen den ce of t h e wild-t ype en zym e.9,41,42 E a r lier com pu t a t ion s pr edict ed t h a t Lys136 a n d Ar g-143 con t r ibu t e m ost t o elect r ost a t ic a t t r a ct ion .13,20 In it ia l in t er pr et a t ion of r esu lt s fr om m ea su r em en t s com bin in g a ct ivit y depen den ce on pH a n d differ en t ion ic st r en gt h s in t h e bovin e en zym e, h owever, su ggest ed t h a t lysin es 122 a n d 136 (h u m a n en zym e n u m ber in g) wer e r espon sible for a ll elect r ost a t ic con t r ol in t h e en zym e’s a ct ivit y.10 F u r t h er wor k on por cin e, ovin e, a n d yea st SODs,43 lysin e m u t a t ion s in sh a r k 44 a n d Xen opu s laevis 45 SODs, m odified bovin e a n d sh a r k SODs,18,46,47 su ppor t ed t h is in t er pr et a t ion , a lt h ou gh t h e possibilit y t h a t
Ar g-143 or a coor din a t ed wa t er con t r ibu t es h a d n ot been r u led ou t .17 Th e la t est in t er pr et a t ion s of t h e pK a effect s in bovin e a n d sh a r k SODs a r e t h a t , in bovin e SOD, t h r ee pK a s con t r ibu t e t o it s obser ved pK a pr ofile—on e a r ou n d 9 a t t r ibu t ed t o su r fa ce lysin es, on e a t 10 a t t r ibu t ed t o su r fa ce a r gin in es, a n d on e a t 11.5 a t t r ibu t ed t o eit h er Ar g-141 (h u m a n 143) or a copper-coor din a t ed wa t er m olecu le.17 Ou r da t a (F ig. 5) sh ow t h a t t h e effect s of Lys-136 m u t a t ion on t h e r ea ct ion r a t e a r e con sider a bly sm a ller t h a n t h ose seen in Ar g-143 m u t a t ion s.20 We fin d t h a t Lys-136, a lt h ou gh in flu en t ia l on en zym e a ct ivit y a n d sequ en ce-con ser ved in m ost Cu ,Zn SODs (a lt h ou gh n ot in va r ia n t , a s eviden ced in ba ct er ia l SODs), is by n o m ea n s a cr u cia l r esidu e. In com pa rin g t h e pH pr ofiles, m u t a t ion of Ar g-143 t o eit h er a n a spa r t a t e or glu t a m a t e r esidu e decr ea sed t h e a ct ivit y by t wo or der s of m a gn it u de, t h e m ost ext r em e effect r esu lt in g fr om sit e m u t a t ion seen t h u s fa r.20 On t h e ot h er h a n d, a sim ila r ch a r ge r ever sa l on m u t a t ion of Lys-136 dr opped t h e a ct ivit y on ly by a bou t h a lf. In t er est in gly, a ll of t h e m u t a n t s of Lys136 h a ve r a t e con st a n t s h igh er t h a n wild-t ype a bove pH 9.5, wh ich is con sist en t wit h a n obser ved pK a a t pH 9.5 bein g du e, in pa r t , t o Lys-136. Th ese differen ces bet ween wild-t ype a n d m u t a n t SODs likely in volve ot h er fa ct or s a s well, h owever, su ch a s su bt le st r u ct u r a l r ea r r a n gem en t s. Th e ion ic st r en gt h depen den cy wa s a lso dom in a t ed by Ar g-143. Ch a r ge n eu t r a liza t ion of Ar g-143 dr ops t h e ion ic st r en gt h depen den cy t o 25% of wild-t ype va lu es in exper im en t s a n d t o 30% in ca lcu la t ion s, wh ile ch a r ge n eu t r a liza t ion of Lys-136 dr ops t h is depen den cy t o 65–75% of wildt ype va lu es. Th e sign ifica n ce of Lys-136 sh ou ld n ot be discou n t ed, h owever. In ba ct er ia l Cu ,Zn SODs, a lt h ou gh t h e sequ en t ia l equ iva len t of Lys-136 h a s been delet ed, it h a s been spa t ia lly r epla ced by a com pen sa t in g lysin e fr om a n ot h er loop r ela t ed by t h e in t er n a l pseu do-t wofold a xis of t h e en zym e.48 Th er efor e, t h e pr esen ce of a lysin e in t h is pa r t icu la r r egion m u st be im por t a n t . Th e decr ea se in a ct ivit y seen a t h igh er pH ca n be a t t r ibu t ed t o t h e gr a du a l depr ot on a t ion of a ll of t h e posit ively ch a r ged gr ou ps in t h e en zym e, a s seen in t h e m u lt iple-sit e t it r a t ion a n a lysis. If on e is t o pin poin t a n y on e r esidu e a s con t r ibu t in g m ost t o in it ia t in g t h e dr op in r a t e a t pH 9.5, h owever, we a gr ee wit h Ba n ci et a l.16 t h a t Ar g-143 is a m u ch m or e likely ca n dida t e t h a n Lys-136, a t lea st in h u m a n SOD. Ou r m u lt iple-sit e t it r a t ion com pu t a t ion s a ssign Ar g-143 a pK a va lu e of a bou t 10.7, m u ch lower t h a n t h e pK a of 12.5 for a st a n da r d a r gin in e. On e m u st , t h er efor e, t a ke in t o a ccou n t t h a t t h e pK a of Ar g-143 is gr ea t ly a ffect ed by t h e r esidu e’s en vir on m en t , n ot su r pr isin g con sider in g it s pr oxim it y t o t h e copper ion . In a ddit ion , t h e in flect ion poin t in t h e pH pr ofile sh ift s dr a m a t ica lly u pon m u t a t ion of Ar g-143 t o lysin e, su ch t h a t t h e declin e in a ct ivit y begin s a t pH 7, a fu ll 2.5 pK u n it s lower !20 Th is dr op in t h e pK a
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wa s r eplica t ed, t h ou gh a ppa r en t ly over est im a t ed, in t h e m u lt iple-sit e t it r a t ion ca lcu la t ion s, wh ich sh owed a decr ea se in pK a fr om 10.7 t o 6.9 on m u t a t ion of Ar g-143 t o lysin e. Mu t a t ion of Lys-136 t o a r gin in e sh ift ed t h e a ppa r en t pK a less t h a n h a lf a pK u n it . Alt h ou gh t h ese r esu lt s do n ot n ecessa r ily r u le ou t t h e possibilit y of t h e t it r a t ion of a copper-bou n d wa t er m olecu le a s t h e sou r ce of t h e a ct ivit y dr op a bove pH 9.5, t h ey do in dica t e t h a t Ar g-143 is m or e likely t o be r espon sible t h a n Lys-136. CON CLU S ION S H u m a n Cu ,Zn SOD depen ds on t h e elect r ost a t ic m a keu p a r ou n d t h e a ct ive sit e ch a n n el t o m a xim ize it s su per oxide t u r n over r a t e. Lys-136, wh ich lies on t h e lip of t h e a ct ive sit e ch a n n el a bou t 10–12 Å fr om t h e copper ion , wor ks in t a n dem wit h Glu -132 a n d Glu -133 t o in flu en ce t h e en zym e’s elect r ost a t ic en vir on m en t a n d bin din g select ivit y. E n zym es wit h m u t a t ion s a t Glu -132, Glu -133 19 a n d Lys-136 a ll sh ow pH pr ofiles a n d ion ic st r en gt h depen den cies con sist en t wit h t h ese r oles. Repla cem en t of Lys-136 wit h eit h er a n eu t r a l or n ega t ively ch a r ged r esidu e decr ea ses t h e r ea ct ion r a t e of t h e en zym e by a bou t a fa ct or of 2, or a bou t t h e sa m e degr ee a s t h e r epla cem en t of Glu -132 wit h glu t a m in e in cr ea ses t h e r a t e.19 In con t r a st , ch a r ge r ever sa l of Ar g-143 h a d a 100fold effect on r ea ct ion r a t e.20 Th e la r ge differ en ce bet ween t h e degr ee of in flu en ce of t h e t r ia d r esidu es a n d Ar g-143 illu st r a t es t h e con t r a st bet ween sim ple elect r ost a t ic effect s a n d t h e m or e com plex elect r ost a t ic a n d m ech a n ist ic in volvem en t of Ar g-143, a n d pr ovides a n est im a t e of t h e con t r ibu t ion of elect r ost a t ics t o r ea ct ion r a t e. In con clu sion , a lt h ou gh Lys-136 is less cr it ica l t o SOD a ct ivit y t h a n Ar g-143, wh ich expla in s it s lower sequ en ce con ser va t ion (u n like t h e sequ en ce-in va r ia n t Ar g-143),40,44,49 Lys-136, a lon g wit h Glu -132 a n d Glu -133, does per for m a n im port a n t elect r ost a t ic r ole in su bst r a t e r ecogn it ion a n d specificit y in Cu ,Zn SOD. ACKN OWLED GMEN TS We t h a n k Mich a el P iqu e a n d Ma lcolm Da vis for h elp in t est in g a n d a lt er in g t h e Br own ia n dyn a m ics pr ogr a m s, Dia n e Ba u er, Ka r in Im la y, a n d Ir m a La r ia for exper t t ech n ica l a ssist a n ce, a n d H a n s P a r ge for cr yst a llogr a ph ic con t r ibu t ion s. Th is wor k wa s su ppor t ed by NIH Gr a n t GM-39345 (J AT, RAH ), GM-37684 (E DG), NSF Gr a n t DIR-882385 (J AT, E DG), NIH F ellowsh ips GM-11612 a n d H L-07695 (CLF ), a post doct or a l fellowsh ip fr om t h e Medica l Resea r ch Cou n cil of Ca n a da (TP L), t h e Ch ir on Cor por a t ion (RAH ), a n d a gr a n t fr om t h e Wellcom e Tr u st (RAH ). Br own ia n dyn a m ics ca lcu la t ion s u sed t h e com pu t er r esou r ces a t Scr ipps a n d a ddit ion a l com pu t er t im e pr ovided by Cr a y Resea r ch , In c. P u lse r a diolysis r a t e st u dies wer e su ppor t ed by NIH Gr a n t GM-23658 a n d con du ct ed a t Br ookh a ven Na t ion a l La bor a t or y, wh ich is oper a t ed u n der con t r a ct DE -
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