Farah Javid-Majd, Michelle A. Stapleton, Marilyn F. Harmon, Brent A. Hanks, Leisha S. Mullins, and Frank M. Raushel*

Transcription

1 14362 Biochemistry 1996, 35, Comprison of the Functionl Differences for the Homologous Residues within the Crboxy Phosphte nd Crbmte Domins of Crbmoyl Phosphte Synthetse Frh Jvid-Mjd, Michelle A. Stpleton, Mrilyn F. Hrmon, Brent A. Hnks, Leish S. Mullins, nd Frnk M. Rushel* Deprtment of Chemistry, Texs A&M UniVersity, College Sttion, Texs ReceiVed My 17, 1996; ReVised Mnuscript ReceiVed August 26, 1996 X ABSTRACT: Crbmoyl phosphte synthetse (CPS) from Escherichi coli ctlyzes the formtion of crbmoyl phosphte from two molecules of MgATP, bicrbonte, nd glutmine. It hs been previously shown tht the mino- nd crboxy-terminl hlves of the lrge subunit of this protein re homologous. A working model for the ctive site structure of the crboxy-terminl domin of the lrge subunit of CPS ws constructed bsed upon mino cid sequence lignments nd the previously determined threedimensionl structures of two mechnisticlly relted proteins, biotin crboxylse nd D-lnine:D-lnine ligse. The model ws tested by muttion of ten mino cid residues predicted to be importnt for binding nd/or ctlysis. The mutted residues were s follows: R571, R675, R715, D753, E761, N827, Q829, E841, N843, nd R845. The mutnt proteins were expressed, purified to homogeneity nd the ctlytic properties determined for vriety of ssy formts. The mutnts E761A, E841Q, N843D, nd R845Q were diminished in their bility to synthesize crbmoyl phosphte. The R715A, Q829A, nd R675A mutnts displyed elevted Michelis constnts for MgADP in the prtil bck rection. The mutnts E761A, N827A, E841Q, N843D, nd R845Q showed significnt increses in the Michelis constnts for either bicrbonte or crbmoyl phosphte. No significnt ltertions were noted upon muttion of either R571 or D753 to n lnine residue nd thus these mino cids do not pper essentil for structure or ctlytic ctivity. These results hve been utilized to further support the proposl tht the C-terminl hlf of the lrge subunit of CPS is primrily responsible for the phosphoryltion of the crbmte intermedite during the finl formtion of crbmoyl phosphte. The mesured effects on the ctlyic ctivities displyed by these muttions were found to be comprble to the previously determined effects fter muttion of the homologous residues locted on the N-terminl hlf of CPS nd lso for those residues mutted within D-lnine:D-lnine ligse [Shi, Y., & Wlsh, C. T. (1995) Biochemistry 34, ]. It is currently believed tht the lrge subunit of crbmoyl phosphte synthetse (CPS) 1 rose from gene dupliction nd fusion of smller ncestrl protein (Nyunoy & Lusty, 1983). This proposl is supported by the high degree of mino cid sequence identity between the mino- nd crboxy-terminl hlves of the lrge subunit of CPS (Nyunoy & Lusty, 1983) nd differentil mutgenesis experiments for residues tht re t or ner the binding sites for ech of the two nucleotides tht re required for the overll synthesis of crbmoyl phosphte (Post et l., 1990). Working models of the ctive sites of the crboxy phosphte nd crbmte domins were constructed bsed upon primry sequence lignments of the two homologous hlves of the lrge subunit of CPS nd two mechnisticlly relted enzymes, biotin crboxylse nd DD-ligse. The working models were further refined by comprison to the X-ry structures of biotin crboxylse (Wldrop et l., 1994) nd DD-ligse (Fn et l., 1994). In the preceding pper (Stpleton et l., 1996) This reserch ws supported in prt by the Ntionl Institutes of Helth (DK-30343) nd the Robert H. Welch Foundtion (A-840). B.A.H. ws supported by n REU wrd to Texs A&M University from the NSF (CHE ). * To whom correspondence my be sent. FAX: (409) E-mil: rushel@tmu.edu. X Abstrct published in AdVnce ACS Abstrcts, October 15, Abbrevitions: CPS, crbmoyl phosphte synthetse; HEPES, N-(2-hydroxyethyl)piperzine-N -2-ethnesulfonic cid; SDS, sodium dodecyl sulfte; PCR, polymerse chin rection; BC, biotin crboxylse; DD-ligse, D-lnine:D-lnine ligse. S (96) CCC: $12.00 we successfully identified number of residues within the crboxy phosphte domin of CPS tht re importnt for the binding of bicrbonte nd the MgATP tht is utilized for the synthesis of the crboxy phosphte intermedite. The experiments reported in this pper re directed towrd the identifiction of those residues tht re responsible for comprble functions within the segment of the ctive site in the crbmte domin of CPS where the second ATP phosphoryltes the crbmte intermedite for the ultimte synthesis of crbmoyl phosphte. Ten residues within the crbmte domin of the lrge subunit of CPS were selected s trgets for mutgenesis. These residues consist of R571, R675, R715, D753, E761, N827, Q829, E841, N843, nd R845. In our preliminry working model, residues R675, R714, D753, nd E761 re proposed to interct directly with the nucleotide while residues Q829, E841, nd N843 re proposed to bind directly to one of the two required metl ions used in the overll rection. Residues R571, N827, nd R845 re proposed to bind to either crbmoyl phosphte or crbmte. These sites hve now been mutted nd the corresponding proteins hve been expressed, purified, nd chrcterized by nlysis of the kinetic constnts for the full biosynthetic rection nd the two prtil rections ctlyzed by CPS. Functionl roles for these residues in the crbmte domin hve now been ssigned bsed upon the observed ctlytic properties of these mutnts in comprison with those 1996 Americn Chemicl Society

2 Mutgenesis of Crbmte Domin of CPS Biochemistry, Vol. 35, No. 45, Tble 1: Primers Used in the Site-Directed Mutgenesis of the Crbmte Domin of CPS plsmid DNA sequence enzyme pms06 5 -GTT AAC CCG CAA GCG GCG-3 E845Q 3 CAA TTG GGC GTT CGC CGC 5 pms08 5 -CTG ATT CAA GTT AAC CCG-3 E841Q 3 -GAC TAA GTT CAA TTG GGC-5 pms09 5 -ATT GAA GTT GAC CCG CGT-3 N843D 3 -TAA CTT CAA CTG GGC GCA-5 pms17 5 -CGT GCA GAA GAC GCT GAA CG-3 R675A 3 -GCA CGT CTT CTG CGA CTT GC-5 pms19 5 -GGT GGT AGC TCC GTC TTA CG-3 R715A 3 -CCA CCA TCG AGG CAG AAT GC-5 pms20 5 -GTT GCT GGC GCA CTT CCT CG-3 D753A 3 -CAA CGA CCG CGT GAA GGA GC-5 pms21 5 -CGC GGT AGC AGT TGA CG-3 E761A 3 -GCG CCA TCG TCA ACT GC-5 pms24 5 -CCT GAT GGC CGT GCA GTT TGC 3 N827A 3 -GGA CTA CCG GCA CGT CAA ACG 5 pms27 5 -CCC GAA CGC TAT CGG TCA GGG 3 R571A 3 -GGG CTT GCG ATA GCC AGT CCC 5 pms29 5 -GAT GAA CGT GGC GTT TGC GG-3 Q829A 3 -CTA CTT GCA CCG CAA ACG CC-5 These mutgenic primers introduced bse chnges t the underlined positions. mutnts constructed t the homologous sites within the crboxy phosphte domin of the lrge subunit. MATERIALS AND METHODS Generl Mterils. The restriction enzymes, regents, nd coupling enzymes were obtined from the suppliers identified in the preceding pper (Stpleton et l., 1996). The bcteril strins nd ssocited plsmids used in this study were lso the sme s those described previously. Construction of the R571A, R675A, R715A, D753A, E761A, N827A, R829A, E841Q, N843D, nd R845Q Mutnts. Site-directed mutgenesis ws performed on the plsmid pms03 using the polymerse chin rection (PCR) nd the overlp extension method of Ho et l. (1989). The pirs of internl oligonucleotides which were used to initite these muttions re listed in Tble 1. A flnking primer with the sequence 5 -CATGGAGAATATTGAGCAGG-3 t position 3291 of the crab gene ws used to mke ll of the mutnts except for R571A. For the R571A muttion, flnking primers with the sequences 5 -CGTGCATTAT- TGCCCCATC-3 t position 2133 nd 5 -CGTTCAGCGTCT- TCTGCACG-3 t position 3650 were used. The 3 -flnking primer used in the cretion of the R675A mutnt ws t position 3785 with the sequence 5 -CGTAAGACGGAGC- TACCACC-3. The 3 -flnking primer used to mke the R715A, D753A, nd E761A mutnts ws locted t position 4111 of the crab gene, nd the sequence of this primer ws 5 -GCAAACTGCACGGCCATCAGG-3. In order to mke the mutnts N827A nd Q829A, the 3 -flnking primer t postion 4392 with the sequence 5 -CCCATGACT- TAATCGGTAGAGCAC-3 ws used. The finl PCR product ws digested with EcoRI nd ScII during the construction of the R571A mutnt. The PCR frgment used to construct the R675A mutnt ws digested with EcoRI nd SphI. To mke the R715A, D753A, nd E761 mutnts, the PCR frgments were digested with SphI nd AflII. The PCR frgments used to mke the N827A, Q285A, E841Q, N843D, nd R845Q mutnts were digested with AflII nd Bpu1102I. The digested frgments were inserted into the pms03 plsmid t the pproprite loctions nd then fully sequenced using the Sequense 2.0 sequencing kit from USB/Amershm. The mutnt plsmids were trnsformed into the RC50 cell line for expression nd subsequent purifiction of the mutnt proteins. Purifiction of Wild-Type nd Mutnt Proteins. The wildtype nd site directed mutnts of CPS were purified ccording to the protocols provided by Mrey et l. (1994) with the exception of the N843D mutnt. The mutnt N843D ws purified using the truncted procedure s described previously for the N301D mutnt of CPS (Stpleton et l., 1996). Kinetic Mesurements. The specific detils for the mesurement of the kinetic prmeters were the sme s those described previously by Stpleton et l. (1996). Crbmoyl phosphte synthesis ws determined by mesuring the rte of citrulline formtion in coupled ssy with ornithine trnscrbmoylse nd ornithine (Snodgrss et l., 1969). The rte of ATP formtion from MgADP nd crbmoyl phosphte ws determined spectrophotometriclly using hexokinse/glucose-6-phosphte dehydrogense coupling system. The rte of ATP hydrolysis ws monitored t 340 nm in the presence or bsence of nitrogen source (NH 3 or glutmine) by coupling the production of ADP to the oxidtion of NADH with the enzymes pyruvte kinse nd lctte dehydrogense. Sttisticl Anlysis of Kinetic Dt. The kinetic prmeters, V m nd V/K m, were determined by fitting the dt to eq 1 with computer progrms obtined from Svnn Shell Softwre. In this eqution V is the initil velocity, K m is the Michelis constnt, nd A is the substrte concentrtion. Nonliner double-reciprocl plots were fit to eq 2. In eq 2, A represents the substrte concentrtion, K 1 nd K 2 re the Michelis constnts t high nd low substrte concentrtion while V 1 nd V 2 re the mximl velocities t high nd low substrte concentrtion. The dt for the enhncement of ATP hydrolysis in the presence of nitrogen source with ll mutnts were fit to eq 3. In this eqution, A is the concentrtion of the nitrogen source (either mmoni or glutmine), V o is the initil enzyme velocity in the bsence of nitrogen source, nd K is the ctivtion constnt for the nitrogen source. The vlue of R defines the rtio of the observed velocities t sturting nd zero levels of either glutmine or mmoni. V ) V m A/(K m + A ) (1) V ) [V 1 A/(K 1 + A)] + [V 2 A/(K 2 + A)] (2) V ) V o (RA + K )/(A + K ) (3) Three-Dimensionl Model of the Crbmte Domin of CPS. The three-dimensionl model of the crbmte domin of CPS ws constructed in the mnner described in the preceding pper for the model of the crboxy phosphte domin. The only difference being in the ssignments of the structurlly conserved regions reltive to those of DDligse (DdlB). The ssigned structurlly conserved regions for the model of the crbmte domin were: (1) DdlB 2-16:CPS , (2) DdlB 30-41:CPS , (3) DdlB 50-63:CPS , (4) DdlB 69-73:CPS , (5) DdlB :CPS , (6) DdlB :CPS , (7) DdlB :CPS , (8) DdlB :CPS , (9)

3 14364 Biochemistry, Vol. 35, No. 45, 1996 Jvid-Mjd et l. Tble 2: Kinetic Prmeters for Rections Ctlyzed by Crbmoyl Phosphte Synthetse Mutnts glutmine-dependent ATPse rection crbmoyl phosphte synthesis rection b enzyme effector c K ATP (µm) V m(µmol/min mg) K HCO3 (mm) V m with Gln (µmol/min mg) V m with mmoni (µmol/min mg) wild-type none ornithine UMP R571A none ornithine UMP R675A none ornithine UMP R715A none ornithine UMP D753A none ornithine UMP E761A none ornithine UMP N827A none ornithine UMP R829A none ornithine UMP E841Q none ornithine UMP N843D none ornithine <0.003 <0.005 UMP R845Q none ornithine <0.005 <0.005 UMP ph 7.6, 25 C, 50 mm HEPES, 100 mm KCl, 20 mm Mg 2+, 50 mm KHCO 3, 5 mm ATP (when KHCO 3 ws the vrible substrte) or 50 mm KHCO 3 (when ATP ws vrible substrte), vrible mounts of glutmine or mmoni. b ph 7.6, 25 C, 50 mm HEPES, 100 mm KCl, 5 mm ATP, 10 mm Orn, 20 mm Mg 2+, 50 mm KHCO 3, either 10 mm of glutmine or 300 mm of NH 4Cl. c Effector concentrtions: ornithine, 10 mm; UMP, 100 µm. DdlB :CPS , (10) DdlB : CPS , (11) DdlB :CPS , (12) DdlB :CPS , (13) DdlB :CPS , (14) DdlB :CPS RESULTS Ten conserved mino cids, locted within the crbmte domin of CPS, were mutted to other residues in order to more fully explore the ctive site(s) of this enzyme nd to compre the functionl differences nd similrities of these specific residues with their homologous counterprts from the crboxy phosphte domin. The residues R571, R675, R715, D753, E761, N827, nd Q829 were chnged to lnine while E841, N843, nd R845 were mutted to glutmine, sprtte, nd glutmine, respectively, using the method of overlp extension PCR. The choice of residues selected for muttion ws bsed on conservtion of these specific sites within the CPS primry sequence from vrious sources nd sequence lignments with the mechnisticlly similr enzymes, biotin crboxylse nd DD-ligse. The mutnt proteins were expressed nd purified to homogeneity s judged by SDS-polycrylmide gel electrophoresis. The effects of these modifictions on the kinetic constnts were determined for ech mutnt by mesuring the full biosynthetic rection nd both of the two prtil rections ctlyzed by CPS in the presence nd bsence of the llosteric effectors, ornithine nd UMP. The kinetic prmeters, K m nd V mx, obtined for the wild-type nd the mutnt enzymes re summrized in Tbles 2-5. A model for the threedimensionl fold of the crbmte domin of CPS ws constructed with the Homology progrm from Biosym, bsed on the X-ry coordintes for DD-ligse (Fn et l., 1994) nd biotin crboxylse (Wldrop et l., 1994) nd is illustrted in Figure 1. The pproximte spcil loctions of the ten residues mutted in this investigtion re indicted by spheres within the R-crbon bckbone of the model. Of the ten residues of CPS tht were mutted in this investigtion there re eight tht hve fully nlogous residues in BC, DD-ligse, nd the crboxy phosphte domin of CPS (N845 of CPS does not hve n nlogous residue in DD-ligse, nd R571 is not conserved in the crboxy phosphte domin of CPS). A superimposition of the R-crbon toms of these eight homologous residues within the structure of DD-ligse nd the model for the crboxy phosphte domin of CPS gives n RMS devition of 3.3 Å. However, the primry sequence lignment of the crbmte domin of CPS with DD-ligse ws slightly different from the one used to crete the model for the crboxy phosphte domin of CPS. In this ltered lignment, R675 of CPS ws not ligned with K97 of DDligse. Removl of R675 from the superimposition gve n RMS devition of only 1.11 Å for the seven remining residues. The re of lowest RMS devition between the BC nd DD-ligse structures ws found in the C-terminl

4 Mutgenesis of Crbmte Domin of CPS Biochemistry, Vol. 35, No. 45, Tble 3: Kinetic Prmeters for the Wild-Type nd Mutnt Enzymes for the ATP Synthesis Rection enzyme effector b (µm) K ADP ATP Synthesis Rection V mx (µmol/min mg) K CP (mm) wild-type none ornithine UMP R571A none ornithine UMP R675A none ornithine UMP nd nd R715A none ornithine UMP D753A none ornithine UMP E761A c none 2400, , 0.02 ornithine 2900, , UMP N827A none ornithine UMP R829A none ornithine UMP E841Q none ornithine UMP N843D none ornithine UMP R845Q none ornithine UMP ph 7.5, 25 C, 20 mm Mg 2+, 100 mm KCl, 20 mm crbmoyl phosphte (when ADP ws the vrible substrte) or 5 mm ADP (when crbmoyl phosphte ws vrible substrte). b When llosteric effector ws present, 10 mm ornithine or 100 µm UMP ws used. c The kinetic dt for the E761A mutnt were fit to eq 3 s described under Mterils nd Methods. Tble 4: Kinetic Prmeters for Bicrbonte-Dependent ATPse Rection enzyme K ATP (µm) K HCO3 (mm) V mx (µmol/min mg) wild-type R571A R675A R715A D753A E761A N827A R829A E841Q N843D R845Q ph 7.5, 25 C, 20 mm Mg 2+, 100 mm KCl, 5 mm ATP (when bicrbonte vried), 50 mm bicrbonte (when ATP vried). domins of these two proteins. Superimposition of the five nlogous resides locted in these sub-domins gve RMS devitions of 0.63 Å between BC nd DD-ligse, 1.09 Å between DD-ligse nd the crbmte domin of the CPS model nd 1.11 Å between BC nd the crbmte domin of the CPS model. The dissimilrities in the structurlly conserved regions nd the primry sequence lignments of DD-ligse with the crboxy phosphte nd crbmte domins of CPS ws the direct cuse for the slightly different Tble 5: Effect of Nitrogen Source on the Glutmine-Dependent ATP Hydrolysis Rection enzyme R for Gln K i for Gln (µm) R for NH 4 + K i for NH 4 + (mm) wild-type R571A R675A R715A D753A E761A ND ND N827A ND ND R829A E841Q ND ND N843D R845Q ph 7.6, 25 C, vrible glutmine or mmonium chloride, 5 mm ATP, 50 mm HEPES, 100 mm KCl, 10 mm ornithine, 20 mm Mg 2+, 50 mm KHCO 3, nd 5 mm ATP. ppernce of the model structure for the crbmte domin of CPS in comprison to the structure for the model of the crboxy phosphte domin. Agin, it must be emphsized tht this model of the crbmte domin of CPS should not be tken s undistorted representtion of the threedimensionl structure of CPS since it ws bsed only on portion of the entire primry sequence. The min purpose of the model ws s n id in the design of muttionl studies. Glutmine- nd Ammoni-Dependent Crbmoyl Phosphte Synthesis. The mutnt enzymes, R571A, R675A, R715A, D753A, E761A, N827A, Q829A, E841Q, N843D, nd R845Q were exmined for their bility to synthesize crbmoyl phosphte using either glutmine or mmoni s nitrogen source. With the exception of R571A, the rtes of crbmoyl phosphte synthesis with either glutmine or mmoni s nitrogen source hve been reduced with ll of these mutnts. Two- to six-fold reductions in the rte of crbmoyl phosphte synthesis were observed for the R675A, R715A, D753 nd R829A mutnts. For the mutnts E761A, N827A, nd E841Q the rte of crbmoyl phosphte synthesis hs been reduced between 10- nd 100-fold while no detectble crbmoyl phosphte synthesis ws found for the N843D nd R845Q mutnts. These results re summrized in Tble 2. ATP Synthesis Rection. The rte of the ATP synthesis rection ws supressed by most of the muttions constructed within the crbmte domin of crbmoyl phosphte synthetse. The most severe losses in the ctlytic ctivity of the prtil bck rection were observed with the N827A, E841Q, N843D, nd R845Q mutnts while smller reductions in rte were observed for the R675, E761A nd N827A mutnts. The pprent K m for MgADP in the bsence of ny llosteric effector ws mesurbly incresed reltive to the wild-type vlue for the R675A, R715A, R829A, nd E841Q muttions. Moreover, the E761A mutnt showed nonliner double-reciprocl plot when the ADP concentrtion ws vried. The lrgest increses in the K m for crbmoyl phosphte were mesured for the E761A, N827A, nd R845Q mutnts. The kinetic constnts re summrized in Tble 3. Bicrbonte-Dependent ATPse ActiVity. For the bicrbonte-dependent ATPse rection, the effects on the ctlytic turnover were in generl rther smll. At one extreme the pprent mximl velocity for the R675A mutnt ws

5 14366 Biochemistry, Vol. 35, No. 45, 1996 Jvid-Mjd et l. FIGURE 1: Model for the three-dimensionl fold of the crbmte domin of CPS from E. coli. This structure ws creted by mpping the CPS mino cid sequence onto the coodintes for the crystl structure of DD-ligse nd biotin crboxylse from E. coli using the Homology progrm from Biosym, Inc. Those residues mutted in this investigtion re indicted by spheres. The figure ws mde with Molscript (Krulis, 1991). reduced slightly while for the E761A nd E841Q mutnts the rte of ATP turnover ws incresed bout 15-fold. However, there were some significnt increses in the pprent K m for bicrbonte. For the E761A, N827A, E841Q, N843D, nd R845Q mutnts, the K m for bicrbonte incresed by t lest n order of mgnitude reltive to the wild-type vlue. Surprisingly, there were lso increses in the Michelis constnt for MgATP for some of the mutnts. The biggest effect ws noted with the E841Q mutnt where the vlue incresed to 100 µm from 7 µm for the wild-type enzyme. All of the kinetic constnts re summrized in Tble 4. Glutmine-Dependent ATP TurnoVer. For the wild-type enzyme, the rte of turnover for ATP increses by t lest n order of mgnitude in the presence of glutmine. For ll of the mutnts exmined in this investigtion, the rte of ADP formtion incresed in the presence of glutmine. The lrgest enhncement ws observed with the R715A mutnt while the smllest effect ws obtined for the E841Q mutnt (Tble 5). A smll elevtion ws observed in the Michelis constnt for MgATP with the R675A nd Q829A mutnts in the bsence of llosteric effectors. Significntly, mny of the mutnts hve higher ffinity for the nucleotide in the presence of UMP. This is in shrp contrst with the effect of this llosteric effector on the wild-type enzyme. These results re summrized in Tbles 2 nd 5. DISCUSSION Ten conserved mino cids locted within the crbmte domin of the lrge subunit of CPS were mutted in n ttempt to identify the roles of these residues during the biosynthesis of crbmoyl phosphte. These prticulr mino cids were trgeted for mutgenesis on the bsis of multiple sequence lignments with the homologous crboxy phosphte domin of CPS nd the mechnisticlly relted proteins, DDligse nd biotin crboxylse. Further insights into the potentil functions for these ten residues were supplied by the X-ry crystl structures for DD-ligse (Fn et l., 1994) nd biotin crboxylse (Wldrop et l., 1994). The mutnt proteins, R571A, R675A, R715A, D753A, E761A, N827A, Q829A, E841Q, N843A, nd R845Q, were expressed nd purified, nd the kinetic constnts for the full biosynthetic rection nd the two prtil rections were quntified using vriety of ssy conditions. In the following nlysis of the observed effects on the kinetic constnts exhibited by these mutttions, we mde the explicit ssumption tht the perturbtions on the bicrbonte-dependent ATPse rection were direct reflection on tht portion of the ctive site involved in the phosphoryltion of bicrbonte. It then follows tht the prtil bck rection (ATP synthetse rection) would serve s reporter for modultions on the other nucleotide site tht is primrily responsible for the phosphoryltion of the crbmte intermedite for the ultimte formtion of crbmoyl phosphte. On the bsis

6 Mutgenesis of Crbmte Domin of CPS Biochemistry, Vol. 35, No. 45, of our previous investigtions, we nticipted tht the mjor effects exhibited by these muttions within the crbmte domin of CPS would primrily, but not exclusively, be found on the prtil bck rection rther thn on the bicrbonte-dependent ATPse rection (Post et l., 1990). This prediction hs been confirmed by nlysis of the experimentl kinetic properties of these specific mutnts. Residues InVolVed in the Binding of Nucleotide. Four of the ten residues investigted in this study were originlly postulted to be ssocited with the binding of the nucleotide used in the phosphoryltion of the crbmte intermedite. One of these residues, D753, ws proposed to interct with the mino group t C-6 of the denine ring. However, exmintion of the kinetic constnts tht pper in Tbles 2-5 indictes tht there re no significnt chnges when this prticulr sprtte is mutted to n lnine residue. Therefore, it ppers unlikely tht the crboxylte side chin of D753 is intercting directly with the denine ring s indicted in the originl model. Two rginine residues, R675 nd R715, were proposed to directly coordinte with the nucleotide. The side chin for R675 ws postulted to ion pir with the β-phosphoryl group of ADP while R715 ws proposed to mke contcts with the R-phosphoryl group nd the nitrogen t N7 of the denine ring. With either of the muttions mde t these positions, there were mesureble increses in the K m for MgADP in the bsence of ny llosteric effectors reltive to the vlue observed for the wildtype enzyme. The remining member of this group, E761, ws proposed to hydrogen bond with the cis-diol of the ribose sugr of the nucleotide. The mutnt constructed t this position ctlyzed brely detectble rte of crbmoyl phosphte synthesis. The bicrbonte-dependent ATPse rection ws enhnced reltive to the wild-type vlue while the prtil bck rection ws chrcterized by nonliner double-reciprocl plots with very slow turnovers. The limiting vlue for the K m of MgADP in the bsence of n llosteric effector (2.4 mm) ws gretly incresed reltive to the wild-type vlue. However, it should be noted tht the K m vlues for both crbmoyl phosphte nd bicrbonte were ech incresed by n order of mgnitude reltive to the vlues obtined for the wild-type enzyme. It therefore ppers tht three of the four residues postulted to interct directly with the nucleotide responsible for the phosphoryltion of crbmte exhibit ctlytic constnts fter mutgenesis consistent with the originl proposl. Residues InVolVed in the Binding of DiVlent Ctions. Three of the originl ten residues (Q829, E841, nd N843) mutted for this investigtion were postulted to be involved in the binding of one of the two divlent ctions needed for this rection. One of these metl ions coordintes the tripolyphosphte moiety of ATP while the other metl serves some other less well-defined function. Thermodynmic binding nd ssocited kinetic studies with the CPS from E. coli hve demonstrted tht this enzyme binds one divlent ction (Mg 2+ or Mn 2+ ) prior to the binding of the first MgATP to the enzyme (Rushel et l., 1979). However, it is uncler whether this divlent ction is required for ech of the three distinct rections ctlyzed by the lrge subunit of CPS. Nevertheless, E841, N843 nd Q829 were postulted to interct directly with one of the two divlent ctions utilized in the crbmte domin of the lrge subunit of CPS in nlogy with the model bsed on the structures of DDligse nd biotin crboxylse. The Q829A mutnt ws ble to synthesize crbmoyl phosphte t respectble rte with moderte increse in the K m for the MgATP nd no significnt chnge in the K m for bicrbonte. However, in the prtil bck rection there ws significnt increse in the K m for MgADP tht substntites the predicted effect on the binding of the metl nucleotide. The other two residues, E841 nd N843, re very sensitive to chnges in the side chin structure. Neither of the mutnts mde t these two positions were ble to mke crbmoyl phosphte t significnt rte nd the prtil bck rection ws eqully diminished. The Michelis constnts for bicrbonte nd crbmoyl phosphte were ll elevted bove the wild-type vlues. Similr result hve been previously observed by Guillou nd Lusty (1992) for n E841K mutnt. Overll, these results re consistent with model for the binding of either of the two metl ions with the phosphoryl groups of both ADP nd crbmoyl-p. Residues tht Interct with Crbmoyl Phosphte. The remining three residues (R571, N827, nd R845) were originlly postulted to constitute portion of the binding site for crbmte or crbmoyl phosphte. The replcement of the rginine t position 845 with glutmine resulted in mutnt tht ws unble to synthesize crbmoyl phosphte nd unble to ctlyze the prtil bck rection. However, the bicrbonte-dependent ATPse rection ws lrgely unffected lthough the K m vlues for bicrbonte nd ATP were elevted. Similr effects on the ctlytic constnts were observed upon muttion of N827. The N827A mutnt hd significnt drop in the bility to synthesize crbmoyl phosphte. The bicrbonte-dependent ATPse rection ws elevted but the ATP synthesis rection ws gretly diminished reltive to the wild-type enzyme. This mutnt enzyme hd the highest K m for crbmoyl phosphte of ny of the mutnts constructed for this investigtion. The remining mutnt, R571A, displyed properties tht were nerly identicl with the wild-type enzyme nd thus it ppers tht this rginine is not importnt for either the mintennce of protein structure or ctlytic ctivity of crbmoyl phosphte synthetse. Working Model for the Crbmte Domin of CPS. In the previous pper, directed t the identifiction of essentil residues within the crboxy phosphte domin of CPS (Stpleton et l., 1996) we constructed working model for the ctive site tht ws bsed upon the known threedimensionl structure for DD-ligse (Fn et l., 1994) nd the ssocited mutgenesis experiments of Shi nd Wlsh (1995). Shown in Figure 2 is n nlogous model for the crbmte domin of CPS refined to incorporte the results obtined in this investigtion. Not included in this model re R571 nd D753 since the kinetic prmeters for the lnine mutnts were lrgely unltered reltive to the wildtype enzyme. The remining eight residues re inserted into the working model bsed upon the sequence lignments with DD-ligse nd biotin crboxylse in ssocition with the known three-dimensionl X-ry structures. The mesured ctlytic properties of the mutnts creted t ech of these positions re fully consistent with the postulted working model. While investigtions of this type cn be effectively utilized to identify criticl residues within the seprte domins it does not pper possible to mke further deductions with regrd to how these puttive functionl domins interct with one nother for the concerted synthesis of crbmoyl phosphte. However, this prticulr feture of

7 14368 Biochemistry, Vol. 35, No. 45, 1996 Jvid-Mjd et l. FIGURE 2: Working model for the interction of the ctive site residues of the crbmte domin of CPS with MgADP nd crbmoyl phosphte. This model ws generted by sequence lignment studies with DD-ligse nd biotin crboxylse in conjunction with the X-ry crystl structures nd the ctlytic properties of the mutnts of CPS constructed in this investigtion. Tble 6: Comprison of Homologous Muttions within DD-Ligse nd the Crboxy Phosphte nd Crbmte Domins of CPS crboxy phosphte domin of CPS crbmte domin of CPS b DD-ligse c mutnt modifiction mutnt modifiction mutnt modifiction R82A no significnt effect on kinetic prmeters K634 not mutted H63Q elevted K m for D-Al 1 R129A elevted K m for ATP in bicrbonte-dependent R675A elevted K m for ADP in prtil bck rection K97 not mutted ATPse rection R169A elevted K m for ATP in bicrbonte-dependent R715A elevted K m for ADP in prtil bck rection K144A K m for ATP incresed ATPse rection D207A no significnt effects on kinetic prmeters D753A no significnt effects on kinetic prmeters E180 not mutted E215A elevted K m for bicrbonte suppressed V mx E761A elevted K m for crbmoyl-p nd bicrbonte E187 not mutted for crbmoyl-p formtion suppressed V mx for crbmoyl-p formtion N283A elevted K m for bicrbonte no crbmoyl-p N827A elevted K m for crbmoyl-p nd bicrbonte R255A no detectble ctivity formtion very low crbmoyl-p formtion Q285A elevted K m for ATP in bicrbonte-dependent Q829A elevted K m for ADP in prtil bck rection D257N no detectble ctivity ATPse rection E299Q elevted K m for bicrbonte low crbmoyl E841Q elevted K m for bicrbonte suppressed V mx E270Q no detectble ctivity phosphte formtion for crbmoyl-p formtion N301D elevted K m for ATP nd bicrbonte low N843D elevted K m for bicrbonte nd crbmoyl-p N272 not mutted crbmoyl-p formtion no crbmoyl-p formtion R303Q elevted K m for bicrbonte suppressed crbmoyl-p formtion R845Q elevted K m for bicrbonte nd crbmoyl-p no crbmoyl-p formtion Stpleton et l. (1996). b This study. c Shi nd Wlsh (1995). the crbmoyl phosphte synthetse rection is wht mkes this enzyme so fscinting. Structurl clues for how this enzyme coodintes the biosynthesis of crbmoyl phosphte must wit completion of the three-dimensionl X-ry stucture (Thoden et l., 1995; Mrin et l., 1995). Comprison of Homologous Muttions in DD-ligse with Those for the Crboxy Phosphte nd Crbmte Domins of CPS. Most of the mutnts constructed nd chrcterized in this investigtion of the crbmte domin of CPS hve comprble sites within the crboxy phosphte domin of CPS nd in the ctive site of DD-ligse [for exmple see Figure 1 of Stpleton et l. (1996)]. Shown in Tble 6 is direct comprison of the most slient effects fter mutgensis of these criticl residues. As expected, the muttions constructed within the crboxy phosphte domin of CPS show mjor perturbtions in the bility of the modified proteins to phosphorylte bicrbonte while homologous muttions in the crbmte domin hve, in generl, specificlly perturbed the bility of the mutnts to phosphorylte crbmte. Moreover, this tble lso indictes tht comprble modultions in the kinetic properties re observed for those residues tht hve been mutted in DD-ligse. It will be quite interesting to determine if mutgenesis of homologous sites in biotin crboxylse show similr properties. It should lso be noted tht mny of the ctive site residues identified in this investigtion hve lso been conserved in the ctive site of glutthione synthetse (Fn et l., 1995). ACKNOWLEDGMENT We thnk Professors Hzel M. Holden nd Grover Wldrop for the X-ry coordintes of biotin crboxylse. REFERENCES Fn, C., Moews, P. C., Wlsh, C. T., & Knox, J. R. (1994) Science 266,

8 Mutgenesis of Crbmte Domin of CPS Biochemistry, Vol. 35, No. 45, Fn, C., Moews, P. C., Shi, Y., Wlsh, C. T., nd Knox, J. R. (1995) Proc. Ntl. Acd. Sci. U.S.A. 92, Guillou, F., Lio, M., Grci-Espn, A., & Lusty, C. J. (1992) Biochemistry 31, Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., & Pese, L. R. (1989) Gene 77, Krulis, P. (1991) J. Appl. Crystllogr. 24, Mrey, S. M., & Rushel, F. M. (1994) Biochemisty 33, Mrin, A., Brvo, J., Fit, I., nd Rubio, V. (1995) Proteins: Struct., Funct., Genet Miles, B. W., Mrey, S. M., Post, L. E., Post, D. J., Chng, S., & Rushel, F. M. (1993) Biochemistry 32, Nyunoy, H., & Lusty, C. J. (1983) Proc. Ntl. Acd. Sci. U.S.A. 80, Post, L. E., Post, D. J., & Rushel, F. M. (1990) J. Biol. Chem. 265, Rushel, F. M., Anderson, P. M., & Villfrnc, J. J. (1978) Biochemistry 17, Rushel, F. M., Rwding, C. J., Anderson, P. M., & Villfrnc, J. J. (1979) Biochemistry 18, Shi, Y., & Wlsh, C. T. (1995) Biochemistry 34, Stpleton, M. A., Jvid-Mjd, F., Hrmon, M. F., Hnks, B. A., J. L. Grhmnn, Mullins, L. S., & Rushel, F. M. (1996) Biochemistry 35, Thoden, J. B., Rushel, F. M., Mrey, S. M., Tomchick, D., & Ryment, I. (1995) Act Crystllogr. D Wldrop, G., Ryment, I., & Holden, H. M. (1994) Biochemistry 33, BI961184Q