Metal-Substrate Interactions Facilitate the Catalytic Activity of the Bacterial Phosphotriesterase

Transcription

1 10904 Biochemistry 1996, 35, Metl-Substrte Interctions Fcilitte the Ctlytic Activity of the Bcteril Phosphotriesterse Suk-Bong Hong nd Frnk M. Rushel* Deprtment of Chemistry, Texs A&M UniVersity, College Sttion, Texs ReceiVed Mrch 18, 1996; ReVised Mnuscript ReceiVed June 3, 1996 X ABSTRACT: The bcteril phosphotriesterse from Pseudomons diminut is zinc metlloenzyme which ctlyzes the hydrolysis of vriety of orgnophosphorus nerve gents with high efficiency. The ctive site of the enzyme consists of coupled binucler metl center embedded within cluster of histidine residues. Potentil protein-substrte interctions t the ctive site were probed by systemtic vrition of metl identity, leving group potentil, phosphte host, nd mino cid replcement. In order to determine the roles of these metl ions in binding nd ctlysis, the microscopic rte constnts nd kinetic prmeters were obtined with vrious divlent ctions. The divlent ctions tht were utilized in this investigtion consisted of Co 2+, Ni 2+, Cd 2+, Zn 2+, Mn 2+, nd the mixed-metl Zn 2+ /Cd 2+ hybrid. The leving group potentil nd phosphte host were vried by ltering the pk of the deprting substituted phenol or thiophenol in either diethyl phosphte or diethyl thiophosphte substrte. The Brφnsted plots for the nonenzymtic hydroxide ctlyzed hydrolysis of these substrtes showed liner dependence between the pseudo-first-order rte constnt nd the pk of the leving group. Enzymtic ctivities of the wild-type enzyme with these sme substrtes vried by over 7 orders of mgnitude over the entire experimentl pk rnge ( ), nd the corresponding Brφnsted plots were nonliner. Those substrtes with leving groups with high pk vlues were limited by the rte of bond clevge while those substrtes hving leving groups with low pk vlues were limited by conformtionl chnge or binding event. Thiophosphte substrtes hving leving groups with high pk vlues were better substrtes thn the corresponding phosphte nlogues. These results re consistent with the direct coordintion of one or both metl ions with the phosphoryl sulfur or oxygen tom of the substrte. A lrge dependence of the rte on the leving group rules out the possibility of protontion of the leving group or electrosttic interction of the leving group oxygen (or sulfur) with metl ion or ctionic group t the ctive site. The lrge differences in the size of the β lg over the rnge of metl ions utilized by the enzyme indicte tht the metl ions polrize the phosphoryl group nd lter the structure of the trnsition stte. The vlues of V/K m for the enzyme-ctlyzed hydrolysis for series of substituted thiophenol nlogues were fold smller thn those obtined for the hydrolysis of the corresponding phenolic substrtes, suggesting tht the bulkier sulfur substituent in the leving group my induce conformtionl restrictions t the ctive site. With the zinc-substituted H201N mutnt enzyme, there ws lrge decrese in the rte of phosphotriester hydrolysis but essentilly no chnge in the rte of thiophosphotriester hydrolysis reltive to the vlues observed for the zinc-substituted wild-type enzyme. These results suggest tht direct perturbtion in the lignd structure of the binucler metl center induces ltertions in the mechnism of substrte hydrolysis. The zinc metlloenzyme phosphotriesterse from Pseudomons diminut is highly efficient ctlyst for the hydrolysis of n extensive vriety of orgnophosphorus nerve gents (Dums et l., 1989; Donrski et l., 1989). Two divlent metl ions re criticl for mximl ctlytic ctivity of the enzyme (Dums et l., 1989; Omburo et l., 1992). The two ntive Zn 2+ ions cn be substituted with either Co 2+, Ni 2+, Cd 2+, or Mn 2+ with the restortion of full ctlytic ctivity. The structure nd mechnism of phosphotriesterse hve been previously investigted using kinetic (Donrski et l., 1989; Cldwell et l., 1991,b), spectroscopic (Lewis et l., 1988; Omburo et l., 1993; Che et l., 1993), genetic This work ws supported by the NIH (GM33894) nd the Robert A. Welch Foundtion (A-840). * To whom correspondence should be ddressed. FAX: (409) E-mil: rushel@tmu.edu. X Abstrct published in AdVnce ACS Abstrcts, August 1, S (96) CCC: $12.00 (Kuo & Rushel, 1994), nd X-ry diffrction methods (Benning et l., 1994, 1995; Vnhooke et l., 1996) in ddition to chemicl modifiction nd inctivtion (Dums & Rushel, 1990; Bnzon et l., 1995,b). From these studies, the hydrolytic rection ctlyzed by phosphotriesterse hs been postulted to proceed vi n S N 2-like ssocitive mechnism with ctivtion of wter molecule by the metl center, followed by the direct hydrolysis of the substrte resulting in n inversion of configurtion t the phosphorus center nd dissocition of products. However, previous studies hve demonstrted tht the electronic chnges in the leving group of the substrtes significntly ffects the ctlytic rectivity of the enzyme (Donrski et l., 1989; Cldwell et l., 1991). Brφnsted plots hve indicted tht the rte-determining step chnges from hydrolysis of the P-O bond to conformtionl chnge nd/or binding events s the pk vlue of the leving group is ltered. The ctive site of this enzyme hs been 1996 Americn Chemicl Society

2 Mechnism of Phosphotriester Hydrolysis Biochemistry, Vol. 35, No. 33, Scheme 1 shown by X-ry crystllogrphy to consist of coupled binucler metl center embedded within cluster of histidine residues (Benning et l., 1995; Vnhooke et l., 1996). The precise roles for ech of the two metl ions in ctlysis remin unknown lthough these metls re believed to ctlyze the hydrolysis of orgnophosphtes by decresing the pk of the ctivted wter molecule nd polrizing the phosphoryl oxygen bond during turnover of the enzyme. The substitution of sulfur for oxygen in orgnophosphorus compounds hs been used in vriety of nonenzymtic nd enzymtic rections to demonstrte the coordintion of metl to sulfur nd oxygen. There re severl reports of studies on rte effects upon substitution of sulfur for oxygen counterprts of phosphotriesters (Ketelr et l., 1952; Fukuto & Metclf, 1956; Heth, 1956,b; Cox & Rmsy, 1964; Murdock & Hopkins, 1968; Fnni et l., 1986; Frschtschi et l., 1990). These investigtions hve demonstrted tht for the simple chemicl hydrolysis of thiophosphtes, the substitution of sulfur tom for the nonbridging phosphoryl oxygen results in slower rte of hydrolysis. However, the phosphorothioltes, with sulfur substitution for bridging oxygen tom, hydrolyze much more rpidly thn the corresponding phosphte nlogues. In rtionlizing the reltive rectivity of sulfur vs oxygen nlogues, polrizbility, electronegtivity, nd lbility of the bridging bond hve been put forth s mjor fctors for these observtions (Cox & Rmsy, 1964; Frey & Smmons, 1985). Recent investigtions hve shown tht the two metl toms bound to phosphotriesterse re in close proximity to one nother nd the wter molecule tht ttcks the phosphoryl center is bound directly to the binucler metl center (Che et l., 1993; Benning et l., 1995; Vnhooke et l., 1996). This report presents detiled investigtion of the potentil protein-substrte interctions between the binucler metl center nd the vilble polrizble sites of the substrte s shown in Scheme 1. These potentil interctions t the ctive site hve now been ddressed by systemtic vrition of metl identity, leving group potentil, nd mino cid replcement. In order to evlute the roles of the binucler metl center, the microscopic rte constnts nd other kinetic prmeters for binding, hydrolysis, nd product dissocition were obtined with vrious metl-substituted enzyme derivtives. In ddition, the effect of substrte structure on ctlytic ctivity ws nlyzed with systemtic vrition of both the leving group nd phosphte host by ltering the pk of the deprting phenol or thiophenol within either diethyl phosphte or diethyl thiophosphte substrte. The very sluggish mutnt enzyme (H201N), substituted with Zn 2+ or Co 2+, ws utilized with the sme series of phosphte nd thiophosphte nlogues to exmine in detil the effects on ctlysis by ltering one of the direct lignds to the binucler metl center. MATERIALS AND METHODS Mterils. Proxon (O,O-diethyl-O-p-nitrophenyl phosphte) ws purchsed from Sigm, nd prthion (O,Odiethyl-O-p-nitrophenyl phosphorothiote) ws obtined from Chem Service. 2- nd 3-fluoro-4-nitrophenols nd 2,6- difluorophenols were from Lncster. Unless noted otherwise, the other chemicls for the syntheses of phosphotriester nlogues were obtined from Aldrich. All solvents used for the syntheses were purified nd dried ccording to published procedures (Perrin & Armrego, 1988). The synthesis of 4-cynothiophenol ws ccomplished by the conversion of 4-cynophenol ccording to the method of Sheley (1978). 4-Mercptobenzmide ws mde from 4-cynothiophenol by slightly modifying the procedure of Hll nd Gisler (1976). A series of proxon nd prthion nlogues nd substituted thiophenol derivtives were prepred s previously described (Donrski et l., 1989; Cldwell et l., 1991), nd further purified by flsh column chromtogrphy. All structures of the synthesized triesters were verified by 1 H NMR nd 13 C NMR spectroscopy; mss spectrl dt were lso consistent with the expected moleculr weights of ll compounds. All buffers were purchsed from Sigm except for N-(2-hydroxyethyl)piperzine-N -2-ethnesulfonic cid (HEPES), 1 which ws obtined from United Sttes Biochemicl. The cell growth, purifiction, nd reconstitution of the wild-type phosphotriesterse with Co 2+, Ni 2+, Cd 2+, Zn 2+, Mn 2+, nd the mixed-metl Zn 2+ /Cd 2+ hybrid, in ddition to the mutnt enzyme, H201N, with Co 2+ nd Zn 2+, were performed s previously described (Omburo et l., 1992; Kuo & Rushel, 1994). Kinetic Mesurements. The pk vlues nd extinction coefficients of the leving group phenols nd thiophenols of the substrtes were obtined from the literture (Donrski et l., 1989; Cldwell et l., 1991) or determined spectrophotometriclly s described previously (Cldwell et l., 1991) using Vrin Cry 2200 spectrophotometer nd re presented in Tbles 1 nd 2. The ctlytic ctivities of metlsubstituted wild-type nd mutnt enzymes were ssyed by routinely mesuring the relese of 4-nitrophenol (ɛ ) M -1 cm -1 ) spectrophotometriclly t 400 nm in 100 mm CHES buffer, ph 9.0, with 1.0 mm proxon s substrte. Rection rtes were determined spectrophotometriclly t 25 C with Gilford Model 260 spectrophotometer. Rtes of chemicl hydrolysis were determined t 25 C by spectrophotometriclly following the relese of the phenol or thiophenol product over 7 hlf-lives t n pproprite wvelength upon ddition of potssium hydroxide to finl concentrtion of 1.0 N. For the enzyme-ctlyzed hydrolysis, the cuvettes contining proxon nlogues ( µm), prthion nlogues ( µm), or phosphorothioltes ( µm) in 100 mm CHES, ph 9.0 or 10.0, were preincubted t 25 C for 10 min. Owing to the limited solubility in wter, ll thiophosphtes or phosphorothioltes were tested s substrtes in 15 or 20% methnol/wter mixtures, respectively (Donrski et l., 1989). Vrious concentrtions of the enzyme were dded to initite the rection. The kinetic prmeters, V mx nd K m, were determined spectrophotometriclly by mesuring the initil rte of product formtion t n pproprite wvelength. 1 Abbrevitions: HEPES, N-(2-hydroxyethyl)piperzine-N -2-ethnesulfonic cid; CHES, 2-(N-cyclohexylmino)ethnesulfonic cid.

3 10906 Biochemistry, Vol. 35, No. 33, 1996 Hong nd Rushel Tble 1: Physicl Constnts of Diethyl-Substituted Phenyl Phosphtes t ph 9 or 10 Tble 2: Physicl Constnts of Diethyl S-p-Substituted Phenyl Phosphorothioltes t ph 9 R ) EtOP(O)(EtO)S- (XVII). b Vlues determined in 10% MeOH. hydrolysis of phosphotriesters, thiophosphotriesters, or phosphorothioltes by 1.0 N KOH were obtined by fit of the dt to eq 5 where A is the concentrtion of the substrte, k is the pseudo-first-order rte constnt, nd t is time. V mx ) k 3 k 5 /(k 3 + k 5 ) (2) V/K m ) k 1 k 3 /(k 2 + k 3 ) (3) log k 3 ) (βpk ) + C (4) A ) A o e -kt (5) R ) EtOP(O)(EtO)O- (XV) or EtOP(S)(EtO)O- (XVI). b Vlues determined t ph 10 (this work). c Vlues tken from Donrski et l. (1989). d Vlues determined t ph 10 (Donrski et l., 1989). e Vlues tken from Cldwell et l. (1991). f Vlues determined t ph 9 (Cldwell et l., 1991). Dt Anlysis. All kinetic dt were nlyzed by fitting to the pproprite equtions with the computer progrms supplied by Svnn Shell Softwre. The vlues of V mx nd K m were determined from fit of the dt to the eq 1 V ) V mx A/(K m + A) (1) where V is the initil velocity, V mx is the mximum velocity, K m is the Michelis constnt, nd A is the substrte concentrtion. In nlyzing the Brφnsted plots, simplified kinetic scheme ws used for the enzymtic hydrolysis of phosphotriesters, nd is presented in Scheme 2, where k 1 nd k 2 re the rte constnts for the formtion of the Michelis complex, k 3 is the rte constnt for the chemicl trnsformtion, nd k 5 is the rte constnt for product dissocition. The Scheme 2 k 1 A k 3 k 5 E y\z EA 98 EPQ 98 E + products k 2 expressions for the kinetic constnts, V mx nd V/K m, cn be described by eqs 2 nd 3, where the microscopic rte constnt, k 3, is dependent on β nd the pk of the leving group s described in eq 4 (Jencks & Crriuolo, 1961). The pseudo-first-order rte constnts obtined for the chemicl RESULTS Chemicl Hydrolysis of Phosphotriester Anlogues. The Brφnsted plots for the hydrolysis of proxon nlogues (XV), prthion nlogues (XVI), nd S-ryl phosphorothioltes (XVII) with 1.0 N KOH show liner dependence between the pseudo-first-order rte constnt nd the pk of the leving group over the whole experimentl rnge of pk vlues s illustrted in Figure 1. It hs been determined tht the chemicl hydrolysis of phosphotriesters is pproximtely n order of mgnitude fster thn tht of thiophosphotriesters. The observed vlues of β lg for the chemicl hydrolysis of thiophenol nd phenol derivtives of phosphotriesters re comprble over the sme pk rnge. The slopes of the lines in Figure 1 give vlues of β lg ) -0.43, -0.35, nd for the hydrolysis of proxon nlogues, prthion nlogues, nd the S-ryl phosphorothioltes, respectively. Hydrolysis Ctlyzed by Zinc-Substituted Wild-Type Enzyme. The vlues of V mx nd K m were mesured for the enzymtic hydrolysis of 13 proxon nlogues, 13 prthion nlogues, nd 7 diethyl S-ryl phosphorothioltes ctlyzed by the zinc-substituted wild-type enzyme t 25 C nd ph 9.0 or 10.0 (Tble 3). The Brφnsted plots of log V mx vs the pk of the leving group phenol in diethyl phosphte or diethyl thiophosphte substrte show nonliner correltions, s illustrted in Figure 2A. The dependence of log (V/K m )

4 Mechnism of Phosphotriester Hydrolysis Biochemistry, Vol. 35, No. 33, FIGURE 1: Brφnsted plots of log k versus the pk of the leving group for the hydroxide-ctlyzed hydrolysis of series of phosphtes (b), thiophosphtes (O), nd phosphorothioltes (4). The respective β lg vlues re -0.43, -0.35, nd on the pk of the leving group lso follows similr nonliner Brφnsted-type reltionship (figure not shown). The liner segments of ech Brφnsted plot intersect t pk of pproximtely 8.0 for the hydrolysis of proxon nlogues nd 8.5 for the hydrolysis of the prthion nlogues with this enzyme. Those substrtes with leving groups in the lower rnge of pk vlues re hydrolyzed t the sme rte while those substrtes in the higher rnge of pk vlues show n enhnced sensitivity to the specific substituent on the leving group. The Brφnsted-type reltionships shown in Figure 2A give lrge negtive vlues of β lg of -2.2, -2.0, nd -1.0 for the rections of zinc-substituted wild-type enzyme with phosphtes, thiophosphtes, nd phosphorothioltes, respectively, nd the dt re presented in Tble 3. Hydrolysis Ctlyzed by Cdmium-Substituted Wild-Type Enzyme. The plots of log V mx versus the pk of the deprting group phenol or thiophenol for rections of the cdmium-substituted wild-type enzyme with substrtes disply nonliner Brφnsted correltions (Figure 2B). Nonliner Brφnsted plots of log (V/K m ) vs the pk of the leving group were lso observed (figure not shown). The crossover pk vlues re 8.1 nd 8.5 for the phosphte nd thiophosphte derivtives, respectively. The smll dependence on log V mx for the ctlytic hydrolysis of thiophosphtes by the cdmiumsubstituted wild-type enzyme with incresing pk of the leving group (β lg ) -1.4) significntly differs from the lrge dependence on the pk of the leving group for rections of phosphtes with the sme enzyme, where the vlue for β lg ) The Brφnsted slope for the enzymtic hydrolysis of the phosphorothiolte substrtes gives vlue of β lg ) The dt re presented in Tble 4. Hydrolysis Ctlyzed by Mngnese-Substituted Wild-Type Enzyme. The nonliner Brφnsted plots of log V mx vs the pk of the leving group for ctlysis of the hydrolysis of phosphtes nd thiophosphtes by the mngnese-substituted wild-type enzyme re shown in Figure 2C. The Brφnsted plots of log (V/K m ) versus the pk of the leving group for rection of the mngnese-substituted wild-type enzyme with phosphtes nd thiophosphtes hve nonliner correltions (figure not shown). The crossover points from the Brφnsted plots re found t pk vlues of 8.2 nd 9.1 for phosphtes nd thiophosphtes, respectively. The thiophosphte series gives lrge negtive vlue of β lg ) -4.3 compre to vlue of β lg ) -3.2 for the proxon nlogues. Identicl experiments were lso conducted with the nickel- nd cobltsubstituted phosphotriesterse for the series of proxon nd prthion nlogues, nd the zinc/cdmium hybrid enzyme ws tested in the sme mnner (figures not shown). Hydrolysis Ctlyzed by Mutnt H201N Enzyme. The rection of substrtes with the mutnt enzyme (H201N) reconstituted with either Co 2+ or Zn 2+ lso shows nonliner Brφnsted-type reltionships of log V mx versus the pk of the leving group s illustrted in Figure 2D. The vlues of V mx nd V/K m for the hydrolysis of the series of phosphotriesters by the zinc-substituted mutnt re smller thn those mesured for the rection ctlyzed by the wild-type enzyme with these sme substrtes. However, the rte constnts for the hydrolysis of the thiophosphte triesters re nerly the sme for the zinc-substituted wild-type protein nd the H201N mutnt. Clcultion of Rte Constnts for the Kinetic Model. The nonliner Brφnsted plots for V mx nd V/K m with ll eight metl derivtives of phosphotriesterse were fit to the kinetic model tht ppers in Scheme 2. The microscopic rte constnts for the binding (k 1 ) nd dissocition (k 2 ) of the substrte to the Michelis complex in ddition to the rte constnts for the dissocition of the enzyme-product complex (k 5 ) nd the β lg vlues re listed in Tble 5. DISCUSSION The Brφnsted nlysis for the phosphotriesterse-ctlyzed hydrolysis of proxon nlogues exhibited biphsic dependence on the vlues for V mx nd V/K m with the pk of the leving group (Cldwell et l., 1991). The independence on the enzymtic rte of hydrolysis for those substrtes with leving groups hving pk vlues of <8 ws in significnt contrst to the rther lrge dependence (β lg ) -1.8) on the kinetic constnts found with those nlogues with leving groups hving pk vlues >8. Cldwell et l. (1991) demonstrted tht the ctul bond clevge step (k 3 ) limited the overll rte for the enzymtic hydrolysis when the pk of the leving group ws >8, wheres the rte-limiting step ws n ssocited conformtionl chnge or diffusioncontrolled dissocition when the pk vlue of the leving group ws <8. These results indicted tht the ltertion in the effective chrge on the leving group oxygen resulted in chnge in the rte-limiting step. In contrst, the Brφnsted-type correltion for log k hyd with the pk of the leving group for the hydroxide ctlyzed hydrolysis of these sme substrtes showed strictly liner reltionship with β lg ) throughout the whole pk rnge. The present investigtion hs now included dditionl substrtes with leving groups tht further extend the originl pk rnge from high of 8.5 to Corresponding Brφnsted plots for the hydrolysis of these substrtes by the phosphotriesterse reconstituted with either Zn 2+, Co 2+, Cd 2+, Ni 2+, Mn 2+, or the mixed-metl Zn 2+ /Cd 2+ hybrid were obtined nd the microscopic rte constnts clculted. Moreover, two dditionl sets of substrte nlogues were synthesized by substitution of sulfur for n oxygen tom in either the nonbridging or the bridging phosphoryl positions of the

5 10908 Biochemistry, Vol. 35, No. 33, 1996 Hong nd Rushel Tble 3: Kinetic Constnts for the Zn/Zn-Phosphotriesterse R ) EtOP(O)(EtO)O- (XV), EtOP(S)(EtO)O- (XVI), or EtOP(O)(EtO)S- (XVII). proxon nlogues. This ws done to probe for specific metl-substrte interctions in the trnsition stte with the wild-type nd H201N mutnt enzymes substituted with vrious divlent metl ions. Trnsition-Stte Structure for Chemicl Hydrolysis of Phosphotriester Anlogues. The liner free energy reltionships for the chemicl hydrolysis of phosphte, thiophosphte, nd phosphorothiolte triesters re presented in Figure 1. The slopes from these Brφnsted plots reflect the chrge ssocited with the leving group in the trnsition stte structure nd re directly relted to the degree of bond formtion nd clevge (Jencks & Gilchrist, 1968). The results obtined here re therefore consistent with very similr trnsition-stte structures for ech of the three series of triester nlogues. However, the vlue of β lg obtined for the thiophosphte triesters is less negtive thn those obtined for the series of phosphte nd phosphorothiolte nlogues. This difference indictes tht the extent of phenolic oxygen bond clevge with the thiophosphte triesters in the trnsition stte is less developed thn the extent of bond clevge in the other two series of substrtes. Moreover, when sulfur is substituted for the phosphoryl oxygen (PdO), the corresponding triesters re hydrolyzed by KOH bout n order of mgnitude more slowly. This result is consistent with the reduced electronegtivity for sulfur (reltive to oxygen) tht diminishes the electrophilic chrcter of the phosphorus center nd thus the rte of hydrolysis for the thiophosphtes decreses ccordingly. Substitution of sulfur for the bridging oxygen hs the opposite effect since the rte of hydrolysis for the phosphorothiolte series is pproximtely n order of mgnitude fster thn tht observed for the phosphte triesters. This increse in rectivity, however, cn be directly ttributed to the increse in the cidity of the thiophenol leving group reltive to the prent phenol. When the rectivities re compred bsed on the pk of the leving group, the rte constnts for hydrolysis re essentilly the sme. Potentil Roles of Metl Ions in Binding nd Ctlysis. Phosphotriesterse requires two divlent metl ions for enzymtic ctivity. Model studies show tht metl ions cn effectively contribute to n increse in the rte of hydrolysis of phosphte triesters (Ketelr et l., 1956; Mortlnd & Rmn, 1967; Gellmn et l., 1986; Menger et l., 1987; Breslow & Singh, 1988; Morrow & Trogler, 1989; Shrm et l., 1990; Hy & Govn, 1990; Vitrius & Sulttos, 1995). Possible roles of metl ions in these studies include the following: (1) The metl ions my decrese the pk of the bound wter molecule nd increse the nucleophilic chrcter of the ttcking hydroxide. (2) The metl ions cn increse the polriztion of the PdO (or PdS) bond, nd thereby

6 Mechnism of Phosphotriester Hydrolysis Biochemistry, Vol. 35, No. 33, FIGURE 2: Brφnsted plots for the dependence of log V mx on the pk of the leving group. (A-C) Brφnsted plots for the hydrolysis of series of phosphtes (b), thiophosphtes (O), nd phosphorothioltes (2) ctlyzed by the Zn 2+ -substituted phosphotriesterse (A), Cd 2+ -substituted phosphotriesterse (B), nd Mn 2+ -substituted phosphotriesterse (C). (D) Brφnsted plots of log V mx for the hydrolysis of series of phosphtes (b), nd thiophosphtes (O) by the Zn 2+ -substituted H201N mutnt enzyme (the solid lines), nd series of phosphtes (2), nd thiophosphtes (4) by the Co 2+ - substituted H201N mutnt enzyme (the dshed lines) versus the pk of the leving group. ccelerte the pproch of n ttcking hydroxyl ion by incresing the electrophilic chrcter of the phosphorus center. (3) The metl ions my neutrlize the development of negtive chrge on the leving group. All three contributions my operte in the functioning of phosphotriesterse. The ph-rte profiles for the wild-type phosphotriesterse reconstituted with Zn 2+, Co 2+, Ni 2+, Mn 2+, nd Cd 2+ ll indicte tht single ionizble group from the enzyme must be unprotonted for enzymtic ctivity. However, the kinetic pk vlue is the lowest (pk ) 5.8) with the zinc-substituted enzyme nd the highest (pk ) 8.1) with the cdmiumsubstituted enzyme (Omburo et l., 1992). These results re consistent with the direct ligtion of the hydrolytic wter molecule (or hydroxide) with one or both of the metl ions in the ctive site. 2 The X-ry crystl structures of the Cd 2+ - nd Zn 2+ -substituted enzymes show solvent wter molecule (or hydroxide) bridging the two metl ions (Benning et l., 1995; Vnhooke et l., 1996). An obvious generl bse hs not been identified. To dte, there hs been no evidence to support the chrge neutrliztion of the leving group during the hydrolysis of phosphotriester substrtes by the enzyme since there is no loss of ctivity t high ph (potentil proton 2 The pk vlues of the metl-bound wter molecule in complexes of Zn 2+, Co 2+, Cd 2+, Ni 2+, nd Mn 2+ re 8.8, 8.9, 9.0, 10.6, nd 10.6, respectively (Bsolo & Person, 1967), wheres the pk vlues of crbonic nhydrse contining Zn 2+, Co 2+, Cd 2+, nd Mn 2+ re 6.9, 6.8, 9.1, nd 8.2, respectively (Lindskog, 1982). Similrly, the pk vlues of the metl-substituted phosphotriesterse determined by kinetic methods re 5.8, 6.5, 8.1, 7.4, nd 7.0 for Zn 2+, Co 2+, Cd 2+, Ni 2+, nd Mn 2+ -enzyme (Omburo et l., 1992). The differences in the observed pk vlues between the metl-wter complexes nd the metl-substituted enzymes re likely due to chnges in the coordintion number nd ltertions in the geometry of the proteinmetl complexes. donor) nd the β vlues for the hydrolysis of triesters with vrious leving groups re so lrge (> 2). Therefore, the focus of this investigtion is directed t whether one or both metl ions in the binucler metl center of phosphotriesterse re directly involved in the polriztion of the PdO or PdS bond during ctlysis. Structure-RectiVity Reltionships in Enzymtic Hydrolysis of Substrtes by Wild-Type Phosphotriesterse. Brφnsted plots for the hydrolysis of substrtes by the Zn 2+ /Zn 2+ - phosphotriesterse in Figure 2A indicte tht the rte-limiting step chnges from hydrolysis of the P-O bond to conformtionl chnge or binding event s the pk vlue of the leving group for the substituted phosphtes nd thiophosphtes is lowered. The β lg for V mx is -2.2 for the series of proxon nlogues with pk vlues >7.9 nd -2.0 for the series of thiophosphte nlogues with pk vlues >8.5. The lrge negtive β lg for the enzymtic rections strongly suggests tht the chemicl step is rte-limiting for phosphte nd thiophosphte nlogues with elevted pk vlues. The rther lrge dependence on the rte with the leving group bility of the product would pper to exclude the possibility of direct electrosttic interction with the binucler metl center or proton donor t the ctive site. In ddition, these high β lg vlues strongly suggest tht the trnsition stte of the enzymtic rection with substrtes hving poor leving groups is significntly dissocitive nd quite product-like, implying very lte trnsition stte. Figure 2A lso shows tht the thiophosphte nlogues re enzymticlly hydrolyzed significntly fster thn the corresponding phosphte nlogues s the pk of the leving group becomes > 8. The hydroxide-ctlyzed rections show the opposite trend. This observtion supports the proposl tht one or both of the metl ions in the binucler metl center directly coordintes the phosphoryl group by polriztion of the PdO (or PdS) bond. It hs been previously demonstrted tht the Cu 2+ -ctlyzed hydrolysis of thiophosphotriesters is much more effective thn with the corresponding oxygen nlogues. For exmple, the hydrolyses of EPN [O-ethyl-O- (p-nitrophenyl)phenyl phosphonothiote] nd prthion proceed 48 nd 20 times fster in the presence of the Cu 2+ ions, respectively, wheres the rte enhncement with proxon under similr conditions is only 2-fold (Ketelr et l., 1956). This observtion cn be explined by the direct coordintion of the metl ions with the sulfur or oxygen tom. This potentil interction is pprently much more effective with sulfur thn with oxygen, nd thus the formtion of this complex gretly increses the electrophilicity of the phosphorus center. Further support for direct cheltion of the phosphoryl group by metl ions t the ctive site is pprent in Figure 2B. The Brφnsted slope of -1.4 for the hydrolysis of thiophosphtes by the Cd 2+ -substituted wild-type enzyme is in shrp contrst to the vlue of -3.0 observed for the corresponding phosphte nlogues. This suggests tht the trnsition stte for the rection of thiophosphtes with the Cd 2+ -substituted enzyme hs more ssocitive chrcter thn tht for the rection with the phosphte nlogues. This rther lrge difference in the size of the β lg probbly rises from the formtion of stronger chelte with the soft sulfur lignd by the soft Cd 2+ within the ctive site. If this explntion is true, then it might be expected tht the β lg vlue for the hydrolysis of the thiophosphte nlogues with the Mn 2+ -substituted enzyme should be very lrge becuse

7 10910 Biochemistry, Vol. 35, No. 33, 1996 Hong nd Rushel Tble 4: Kinetic Constnts for the Cd/Cd-Phosphotriesterse R ) EtOP(O)(EtO)O- (XV), EtOP(S)(EtO)O- (XVI), or EtOP(O)(EtO)S- (XVII). Tble 5: Rte Constnts nd Brφnsted β Coefficients for the Hydrolysis of Phosphotriesters nd Thiophosphotriesters by Metl-Substituted Phosphotriesterse t 25 C metl enzyme substrte k 1 (M -1 s -1 ) k 2 (s -1 ) k 5 (s -1 ) β b c b crossover pk Zn/Zn oxo thio Zn/Zn (H201N) oxo thio Co/Co oxo thio Co/Co (H201N) oxo thio Cd/Cd oxo thio Ni/Ni oxo thio Mn/Mn oxo thio Zn/Cd oxo thio Derived from fit to eqs 2 nd 3. b Derived from fit to eq 4. Mn 2+ is hrd metl ion. In fct, the observed β lg vlue for the Mn 2+ /Mn 2+ -substituted enzyme is If it is ccepted tht the binucler metl center functions, in prt, to polrize the phosphoryl oxygen (or sulfur) bond, then it is now possible to ddress whether one or both metl ions serve in this cpcity. We hve previously demonstrted tht unique hybrid metl derivtive of phosphotriesterse cn be constructed (Omburo et l., 1993). In this hybrid, the binucler metl center is composed of n equl mixture of Zn 2+ nd Cd 2+. In ddition, cdmium NMR hs shown tht ech metl occupies single site in the binucler metl center but, s of yet, we do not

8 Mechnism of Phosphotriester Hydrolysis Biochemistry, Vol. 35, No. 33, know which metl is occupying which site. 3 With the very fst substrtes, proxon nd prthion, the kinetic constnts for the Zn 2+ /Cd 2+ -hybrid re nerly identicl with the mesured kinetic constnts for the Zn 2+ /Zn 2+ -enzyme nd quite distinct from the Cd 2+ /Cd 2+ -enzyme. Thus, when bond clevge is not rte-limiting, it ppers tht the Zn 2+ site in the hybrid is dominting the functionl properties of the hybrid. For those substrtes where the bond clevge step is rtelimiting, mixed results were obtined. With the thiophosphte nlogues, the β lg vlue for the hybrid (-1.6) is intermedite in size reltive to the vlues obtined for the Zn 2+ /Zn 2+ - (-2.0) nd Cd 2+ /Cd 2+ -enzyme (-1.3). However, for the hydrolysis of the phosphte nlogues, the β lg vlue for the hybrid is identicl to the vlue obtined for the Zn 2+ / Zn 2+ -enzyme nd quite different from the Cd 2+ /Cd 2+ - enzyme. Either the complextion scheme is distinctly different in the trnsition stte for the phosphte nd thiophosphte nlogues or else the interreltionship between the closely spced metls in the ctive site is more complicted thn this simple model would suggest. In the bsence of ny further experimentl evidence, our working model will depict polriztion of the phosphoryl bond by both metl ions in the binucler metl center. Further refinement of this model will hve to wit dditionl crystlliztion nd/or spectroscopic experiments in the presence of substrte nlogues. Hydrolysis of Substrtes by the H201N Mutnt Phosphotriesterse. The histidine t position 201 ws originlly mutted to n sprgine in n ttempt to determine whether the imidzole side chin ws prticipting in the functioning of the phosphotriesterse either s lignd to the metl center or, lterntively, s n ctive-site bse. Muttion of this residue results in the loss of ctlytic ctivity, nd the X-ry crystl nlysis by Benning et l. (1995) clerly indictes tht this residue is lignd to the more solvent-exposed metl within the binucler metl center. We initilly nticipted tht the Brφnsted plots for this mutnt would be similr in shpe to those observed for the wild-type enzyme except tht the crossover point for those substrtes tht would be limited by conformtionl chnges or binding events would occur t much lower pk vlue for the leving group. To certin extent, this is true for the effect on V mx nd V/K m with the pk of the leving group for the hydrolysis of the phosphte esters, but it is not true for the hydrolysis of the thiophosphte esters. With the phosphte esters, there lso ppers to be generl reduction in ll of the clculted rte constnts reltive to the wild-type enzyme. And thus the mximlly obtinble vlue for k ct is lower for the mutnt even with substrtes hving leving groups of very low pk vlues. It is lso interesting to note tht the rtes of thiophosphte hydrolysis by the zinc-substituted enzyme re nerly identicl to the wild-type vlues wheres the rtes of phosphte hydrolysis re significntly depressed. We nticipte tht this perturbtion in the structure of the binucler metl center hs not only perturbed the ctlytic efficiency of the enzyme but lso ltered conformtionl chnges tht re required for substrte binding nd/or product relese. 3 We suspect tht zinc is ligted to the more buried His-55, His-57, nd Asp-301 while cdmium is ligted to His-201 nd His-230, but confirmtion of this proposl will hve to wit further crystllogrphic or NMR experiments. FIGURE 3: Working model for the hydrolysis of substrtes by the bcteril phosphotriesterse. The metl ions re shown s blck spheres nd coordintion bonds s dshed lines. Mechnism of Hydrolysis. 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