Use of Site-D irected M utagenesis to Investiga te the Properties of Recom binan t H irud in-hv2

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1 14 1 V o l114,n o Ch inese Jou rnal of B iochem istry and M o lecu lar B io logy Feb 1998 (, ),,, rhv rhv 2 Gln11 A sn47 H is11 L ys47, rhv 22H 11 30%, rhv 22K47 61% K i, rhv 22 H 11 K i 14, rhv 22K47 K i 14,, rhv 22H 11, : 47 L ys 11 :,,, Use of Site-D irected M utagenesis to Investiga te the Properties of Recom binan t H irud in-hv2 : , : L u Yu2Feng W ang Zhao2H u i H an Yu2M in (Institu te of B iop hy sics A cad em ia S inica, B eij ing ) Abstract H irudin HV 2 is a po ten t and h igh ly specific inh ib ito r of th rom b in T he purpo se of th is research w as to study the k inetics of com p lex fo rm ation betw een recom b inan t h irudin (rhv 2) o r recom b inan t h irudin m u tan ts and th rom b in In o rder to elucidate the reactive site of the inh ib ito r, p redeterm ined am ino acid sub stitu tion s w ere in troduced by m ean s of site2di2 rected m u tagenesis and random m u tagenesis of a h irudin gene T he an tiam ido lytic activity, b ioactivity and stab ility of rhv 2 and its tw o m u tan ts w ere com pared In com parison w ith re2 com b inan t h irudin (K i= m o lgl ), m u tan t rhv 22K47 show ed a low er K i value ( m o lgl ) and m u tan t rhv 22H 11 show ed a h igher K i value ( m o lgl ) B ioac2 tivity of m u tan t rhv 22K47 increased by 61% and tho se of m u tan t rhv 22H 11 decreased by 30% T he stab ility of recom b inan t h irudin and its m u tan ts w ere tested at variou s ex trem e condition ṡ In com parison w ith recom b inan t h irudin, the increase in stab ility of the tw o m u2 tan ts w ere ob served w hen the alkaline so lu tion w as heated at 70 T here w as a decrease in stab ility of rhv 22H 11 w hen the acidic o r alkaline so lu tion w as incubated at 25 fo r 2 hou rṡ A cco rding to above resu lts, the structu re of h irudin and its in teraction w ith th rom b in w as af2 fected by bo th the hydrogen bond and static charge of L ys 47 It also suggested that Gln 11 m ay be ano ther im po rtan t am ino acid in h irudin 32

2 Key words: r2h irud in, Gene m u tagenesis, K inet ics, Stab ility [ 1, ], DNA 65 66, [ 2 ] [ 3, 4 ] : 3 N (1 52 ) C (53 65 ) 1 1 N, C, P ro462l ys472p ro48, N [ 5 ], [ 6 ],,,,, rhv 2 Ch rom ozym TH (To s2gly2p ro2a rg2n an ) Boeh ringer pya 2C, JM 109, 2 pjh 158 N ew England B io2 lab s P rom ega [Α2 35 S ]2dA T P ( C igmm o l) D u Pon t : P rom ega A ltered Sites TM DNA : Pharm acia T 7DNA : pya 2C pselect2hv 2 H indg Kpng, pya 2C psleclect2hv 2 HV 2, T 4DNA, pya 2C2HV 2 pjh 158 Bam H g, HV 2 pjh 158, pjh 1582HV 2, JM 109 [ : L o ison ],, YEPD : m l, [8 ] : Chang [ 6 ] Ch ro2 m ozym TH ( ) M arkw ardt [ 1 ] : [10 ] (A GA CTC T GG CT T T GG A GT G) HV 2 pselect2 DNA, DNA (pselect2hv 2), JM 109, DNA 33

3 , L ys47 H is rhv2 BJ219952SC, 50 m l YEPD, 30, 5% m l, h 213 rhv2, D EA E2SephadexA 225, SephadexG225,, U ltrashere2od S D EA E, 95% 214 rhv2 rhv 2 3, T ab le 1 rhv 22H 11 rhv 2 70%, rhv 22K47 rhv 2 161% Table 1 B ioactivity of rhv 2 and its m utants Samp le Specific activity 3 gu m g - 1 Relative activity (% w ild type) rhv rhv 22K rhv 22H : A verage value 215 ph F ig 1, (co lum n 1, 2), rhv 22H 11 (co lum n 6) ;, (co lum n 4) rhv 22H 11 (co lum n3, 5) F ig 1 Stability of rhv 2 and its m utants under extrem e ph and temperature Co lum n 1, ph 610, 100 fo r 30 m in, Co lum n 2, ph 610, 100 fo r 60 m in, Co lum n 3, ph 1, 25 fo r 2 h, Co lum n 4, ph 1, 100 fo r 15 m in, Co lum n 5, ph 13, 25 fo r 2 h, Co lum n 6, ph 13, 70 fo r 15 m in 216, (tigh t slow 2 b inding inh ib ition) (F ig 2) : 34

4 E+ S Ω K m ES _ K cat E+ P + I K on K off E I K i= K offgk on ( ) [ I] [E ], : K D = [ I0 ]V SgV 0 - V S (1) K D = K i + K i[s ]gk m (2) [ I0 ], V 0, [V S ], [ K D ] 30 F ig 2 T igh t slow 2binding inh ibition of th rom bin by recom bi2 nant hirudin, [E ],, V S V 0, rhv 22K47 w as p resent at concentrations of 0 (1), 2 (2), 4 (3), 8 (4), 12 (5) and 16 (6) pmo lgl D ata po ints at tim es less than 3 m in have been om itted V 0 V S K D, [ S ] K D, rhv 2, rhv 22H 11 rhv 22K47 K i (F ig 3), m o lgl rhv 22H 11 rhv 2 14, rhv 22K47 rhv 2 14 F ig 3 K i value of recom binant h irudin and its two m utants T he values of K D w ere determ ined as described un2 der 216 T he lines draw n in the figure rep resent the best fit to the equation (2) The unit of K D fo r rhv 22K47, rhv 2 and rhv 22 H 11 are 011, 1 and 10 mmo lgl respectively rhv 22K47; rhv 2; rhv 22H 11 3,, N, ; C, (P1 ), 47 L ys P1, 47 : Glu, Gln A sn,, rhv 2E47, rhv 2Q 47 rhv 2N 47 K i 35

5 rhv 2K47 K i 10 ; Ile, rhv 2I47 K i rhv 2K47 K i rhv 2K47 K i 3 ; A rg L ys, Ε N, rhv 2R 47 rhv 2K47 K i, H is,, rhv 2H 47 K i rhv 2K47 K i [ 5, 11, 12 ],, 47 L ys, 47 [ 13 K i, 47 L ys P1 ], L ys 47 T h r 4,A sp 5 A sn 12,, C 27, 35 47, N, (Cys 6 2Cys 14, Cys 16 2 Cys 28, Cys 22 2Cys 39 ),, Gln 11 H is 11, K i 14,,, Gln H is;,, rhv 22K47 2, rhv 22H 11,,,, rhv 22H 11,, 11,,, References 1 M arkw ardt F H irudin as an inh ibito r M ethod s in E nzym ology, 1970, 19: Do lt J,M uller H P, Selm uller U, Chang J2Y T he comp lete am ino acid sequence of h irudin, a th rom bin specific inh ibito r F EB S L ett, 1984, 165: Fo lkers P J M, C lo re G M, D risco ll P C, Dodt J, Koh ler S, Gronenbo rn A M So lution structure of recom binant h irudin and the L ys247 Glu m utant: A nuclear m agnetic resonance and hybrid distance geom etry 2dynam ical sim ulated anneal2 ing study B iochem istry, 1989, 28: Chang J Y, Stability fo h irudin, a th rom bin2specific inh ibito r J B iol Chem, 1991, 266: Rydel T J, Ravichandran K G, T ulinsky A, Bode W, H uber R, Ro ttsch C, Fenton J W T he structure of a comp lex of re2 com binant h irudin and hum an Α2th rom bin S cience, 1990, 249: Chang J Y T he functional dom ain of h irudin, a th rom bin2specific inh ibito r F EB S L ett, 1983, 164: L o ison G, N guyen2jui lleretm, A louani S,M arquetm P lasm id2transfo rm ed ura3 fur1 double2m utants of S cerevisive: an auto selection system app licable to the p roduction of fo reign p ro tein B iogtechnology, 1986, 4: ,,,, (B ) (Shen Tong2J ian, Ge Q ing2yuan, H an Yu2M in,w ang Zeng2Feng,W ang Q i2song Exp ression of synthesized hirudin gene in yeasṫ S cience in Ch ina (Series B) ), 1991, (8) ,,,, (H an Yu2M in, Ge Q ing2yuan,w ang 36 Zeng2Feng, Guo Yao2Jun, Shen Tong2J ian Purification and identification of recom binant hirudin Chin B iochem J ),

6 1991, 7: ,, (W ang Zhao2H ui, L u Yu2Feng, H an Yu2M in Stability of recom binant h irudin2hv 2 Ch in B iochem J ), 1995, 11: Do lt J, Koh ler S, Baici A Interaction of site specific h irudin variant w ith Α2th rom bin F EB S L ett, 1988, 229: B raun P J,Dennis S, Hofsteenge J, Stone S R U se of site2directed m utagenesis to investigate the basis fo r the specificity of h irudin B iochem istry, 1988, 27: A yala Y M, V indigni A, N ayalm, Spo lar R S, Reco rd M T, J r, Cera E D T hermodynam ic investigation of h irudin bind2 ing to the slow and fast fo rm s of th rom bin: Evidence fo r fo lding transitions in the inh ibito r and p ro tease coup led to bind2 ing J M ol B iol, 1995, 253: