Binuclear Metal Center Research Articles

PII: S0969-2126(01)00700-6

PII: S0969-2126(01)00700-6

Expression, purification and characterization of the soluble CuA domain of cytochrome c oxidase ofParacoccus versutus

The key subunit II of cytochrome c oxidase (CcO) contains a soluble binuclear copper center (CuA) domain. The Cua domain ofParacoccus versutus was cloned, expressed, purified and characterized. The gene encoding the Cua domain in pET11d vector was expressed inE. coli BL21 (DE3). The results showed that the Cua domain was expressed mostly in inclusion bodies and the Cua domain protein synthesized inE. coli cells represents approximately 10 percent of the total cellular proteins. Dissolved in urea, dialyzed and recombined with Cu+/Cu2+ and purified by the Q-sepharose fast flow anion-exchange column and Sephadex G-75 gel filtration column, the soluble purple-colored protein, which shows a single band in electrophoresis, was obtained. The UV-visible absorption spectrum of Cua domain showed that there are intense band at 478 nm and a shoulder peak at 530 nm, and two weak bands at 360 and 806 nm respectively, which can be assigned to the charge transfer and the interactions of obitals of Cu-S and Cu-Cu in the mixed-valence binuclear metal center (Cu2S2R2). The far-UV CD spectrum indicated that this domain is predominantly in β-sheet structure. The fluorescence spectra showed that its maximal excitation wavelength and maximal emission wavelength are at 280 and 345 nm, respectively.

Arabidopsis Glyoxalase II Contains a Zinc/Iron Binuclear Metal Center That Is Essential for Substrate Binding and Catalysis

Glyoxalase II participates in the cellular detoxification of cytotoxic and mutagenic 2-oxoaldehydes. Because of its role in chemical detoxification, glyoxalase II has been studied as a potential anti-cancer and/or anti-protozoal target; however, very little is known about the active site and reaction mechanism of this important enzyme. To characterize the active site and kinetic mechanism of the enzyme, a detailed mutational study of Arabidopsis glyoxalase II was conducted. Data presented here demonstrate for the first time that the cytoplasmic form of Arabidopsis glyoxalase II contains an iron-zinc binuclear metal center that is essential for activity. Both metals participate in substrate binding, transition state stabilization, and the hydrolysis reaction. Subtle alterations in the geometry and/or electrostatics of the binuclear center have profound effects on the activity of the enzyme. Additional residues important in substrate binding have also been identified. An overall reaction mechanism for glyoxalase II is proposed based on the mutational and kinetic data from this study and crystallographic data on human glyoxalase II. Information presented here provides new insights into the active site and reaction mechanism of glyoxalase II that can be used for the rational design of glyoxalase II inhibitors.

Isolation and characterization of an unusual 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4.

A genomic library of the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-containing plant growth-promoting bacterium Enterobacter cloacae UW4 in pUC19 in Escherichia coli was screened for the ability to utilize ACC as a sole source of nitrogen. One of the clones that was isolated contained a plasmid with an insert of approximately 0.8 kb that conferred ACC deaminase activity. Sequence analysis revealed that this DNA fragment contains an open-reading frame of 696 nucleotides predicted to encode a protein of 232 amino acids, a member of the amidohydrolase protein superfamily, i.e., a deaminase that contains a mononuclear or binuclear metal center as compared to the canonical ACC deaminase which contains pyridoxal phosphate as a co-factor.

Sensing electrons: protein phosphatase redox regulation

Regulation of signal transduction is a multifaceted process involving ligands, receptors, second messengers, protein kinases and phosphatases, transcription factors, and target enzymes and genes. Here we consider how oxidative stress and cellular redox can provide additional mechanisms for gating signal transduction. The activities of both protein serine/threonine and tyrosine phosphatases are affected by the oxidation state of redox-sensitive functional groups. For the phosphatases calcineurin and purple acid phosphatase, activity depends upon the oxidation state of an active-site Fe ion. 1. Yu L. et al. Evidence that calcineurin accommodates an active site binuclear metal center. J. Am. Chem. Soc. 1995; 117: 10147-10148 Crossref Scopus (59) Google Scholar , 2. Yu L. et al. Spectroscopic and enzymatic characterization of the active site dinuclear metal center of calcineurin: implications for a mechanistic role. Biochemistry. 1997; 36: 10727-10734 Crossref PubMed Scopus (55) Google Scholar , 3. Merkx M. et al. The activity of oxidized bovine spleen purple acid phosphatase is due to an Fe(III)Zn(II) ‘impurity’. Biochemistry. 1998; 37: 11223-11231 Crossref PubMed Scopus (40) Google Scholar In the tyrosine phosphatases, oxidation of an active-site cysteine residue leads to inactivation. 4. Denu J.M. et al. Protein tyrosine phosphatases: mechanisms of catalysis and regulation. Curr. Opin. Chem. Biol. 1998; 2: 633-641 Crossref PubMed Scopus (334) Google Scholar , 5. Denu J.M. et al. Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation. Biochemistry. 1998; 37: 5633-5642 Crossref PubMed Scopus (816) Google Scholar Redox reactions involving these functional groups and cellular oxidants and reductants could provide a direct link between phosphatase activity and cellular redox perturbations. We propose that protein phosphatases could be key mediators for the regulation of signal transduction by oxidative stress or cytoplasmic redox, or both.

Self-assembly of the binuclear metal center of phosphotriesterase.

The active site of the bacterial phosphotriesterase (PTE) from Pseudomonas diminuta contains two divalent metal ions and a carboxylated lysine residue. The native enzyme contains two Zn(2+) ions, which can be replaced with Co(2+), Cd(2+), Ni(2+), or Mn(2+) without loss of catalytic activity. Carbon dioxide reacts with the side chain of lysine-169 to form a carbamate functional group within the active site, which then serves as a bridging ligand to the two metal ions. The activation of apo-PTE using variable concentrations of divalent metal ions and bicarbonate was measured in order to establish the mechanism by which the active site of PTE is self-assembled. The time courses for the activation of apo-PTE are pseudo-first-order, and the observed rate constants are directly proportional to the concentration of bicarbonate. In contrast, the apparent rate constants for the activation of apo-PTE decrease as the concentrations of the divalent cations are increased and then become constant at higher concentrations of the divalent metal ions. These results are consistent with a largely ordered kinetic mechanism for the assembly of the binuclear metal center where CO(2)/bicarbonate reacts with the apo-PTE prior to the binding of the two metal ions. When apo-PTE is titrated with 0-8 equiv of Co(2+), Cd(2+), or Zn(2+), the concentration of activated enzyme increases linearly until 2 equiv of metal ion is added and then remains constant at elevated levels of the divalent cations. These results are consistent with the synergistic binding of the two metal ions to the active site, and thus the second metal ion binds more tightly to the protein than does the first metal ion. Measurement of the mean dissociation constant indicates that metal binding to the binuclear metal center is strong [(K(alpha)K(beta))(1/2) = 6.0 x 10(-)(11) M and k(off) = 1.5 x 10(-)(3) min(-)(1) for Zn(2+)]. The removal of the carbamate bridge through the mutagenesis of Lys-169 demonstrates that the carbamate bridge is required for both efficient catalysis and overall stability of the metal center.

Phosphotriesterase: an enzyme in search of its natural substrate.

The bacterial PTE is able to catalyze the hydrolysis of a wide range of organophosphate nerve agents. The active site has been shown to consist of a unique binuclear metal center that has evolved to deliver hydroxide to the site of bond cleavage. The reaction rate for the hydrolysis of activated substrates such as paraoxon is limited by product release or an associated protein conformational change.

Crystallization and preliminary X-ray diffraction data for a purple acid phosphatase from sweet potato.

Purple acid phosphatase from sweet potato is a homodimer of 110 kDa. Two forms of the enzyme have been characterized. One contains an Fe-Zn centre similar to that previously reported for red kidney bean purple acid phosphatase. Another isoform, the subject of this work, is the first confirmed example of an Fe-Mn-containing enzyme. Crystals of this protein have been grown from PEG 6000. They have unit-cell parameters a = b = 118.4, c = 287.4 A and have the symmetry of space group P6(5)22, with one dimer per asymmetric unit. Diffraction data collected using a conventional X--ray source from a cryocooled crystal extend to 2.90 A resolution. The three-dimensional structure of the enzyme will provide insight into the coordination of this novel binuclear metal centre.

Binuclear Metal Centers in Plant Purple Acid Phosphatases: Fe–Mn in Sweet Potato and Fe–Zn in Soybean

Purple acid phosphatases comprise a family of binuclear metal-containing acid hydrolases, representatives of which have been found in animals, plants, and fungi. The goal of this study was to characterize purple acid phosphatases from sweet potato tubers and soybean seeds and to establish their relationship with the only well-characterized plant purple acid phosphatase, the FeIII–ZnII-containing red kidney bean enzyme. Metal analysis indicated the presence in the purified sweet potato enzyme of 1.0 g-atom of iron, 0.6–0.7 g-atom of manganese, and small amounts of zinc and copper. The soybean enzyme contained 0.8–0.9 g-atom of iron, 0.7–0.8 g-atom of zinc per subunit, and small amounts of manganese, copper, and magnesium. Both enzymes exhibited visible absorption maxima at 550–560 nm, with molar absorption coefficients of 3200 and 3300 M−1 cm−1, respectively, very similar to the red kidney bean enzyme. Substrate specificities were markedly different from those of the red kidney bean enzyme. A cloning strategy was developed based on N-terminal sequences of the sweet potato and soybean enzymes and short sequences around the conserved metal ligands of the mammalian and red kidney bean enzymes. Three sequences were obtained, one from soybean and two from sweet potato. All three showed extensive sequence identity (>66%) with red kidney bean purple acid phosphatase, and all of the metal ligands were conserved. The combined results establish that these enzymes are binuclear metalloenzymes: Fe–Mn in the sweet potato enzyme and Fe–Zn in soybean. The sweet potato enzyme is the first well-defined example of an Fe–Mn binuclear center in a protein.

Crystal structure of bullfrog M ferritin at 2.8 Å resolution: analysis of subunit interactions and the binuclear metal center

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.

Manganese Is Essential for Catalytic Activity ofEscherichia coliAgmatinase

PurifiedEscherichia coliagmatinase (EC 3.5.3.11) expressed the same activity in the absence or presence of added Mn2+(0-5mM). However, it was strongly inhibited by Co2+, Ni2+, and Zn2+and almost half inactivated by EDTA. Partial inactivation by EDTA yielded enzyme species containing 0.85 ± 0.1 Mn2+/subunit, and it was accompanied by a decrease in intensity of fluorescence emission and a red shift from the emission maximum of 340 nm to 346 nm, indicating the movement of tryptophane residues to a more polar environment. The activity and fluorescence properties of fully activated agmatinase were restored by incubation of dialysed species with Mn2+. Manganese-free species, obtained by treatment with EDTA and guanidinium chloride (3 M), were active only in the presence of added Mn2+. Results obtained, which represent the first demonstration of the essentiality of Mn2+for agmatinase activity, are discussed in connection with a possible binuclear metal center in the enzyme.

A combinatorial library for the binuclear metal center of bacterial phosphotriesterase.

Phosphotriesterase (PTE) is a zinc metalloenzyme that catalyzes the hydrolysis of an extensive array of organophosphate pesticides and mammalian acetylcholinesterase nerve agents. Although the three-dimensional crystal structure of PTE has been solved (M. M. Benning et al., Biochemistry 34:7973-7978, 1995), the precise functions of the individual amino acid residues that interact directly with the substrate at the active site are largely unknown. To construct mutants of PTE with altered specificities for particular target substrates, a simple methodology for generating a library of mutants at specific sites was developed. In this investigation, four of the six protein ligands to the binuclear metal site (His-55, His-57, His-201, and His-230) were targeted for further characterization and investigation. Using the polymerase chain reaction (PCR) protocols, a library of modified PTE genes was generated by simultaneously creating random combinations of histidine and cysteine codons at these four positions. The 16 possible DNA sequences were isolated and confirmed by dideoxy-DNA sequencing. The 16 mutant proteins were expressed in Escherichia coli and grown with the presence or absence of 1 mM CoCl2, ZnSO4, or CdSO4 in the growth medium. When grown in the presence of CoCl2, the H57C protein cell lysate showed greater activity for the hydrolysis of paraoxon than the wild type PTE cell lysate. H201C and H230C exhibited up to 15% of the wild-type activity, while H55C, a green protein, was inactive under all assay conditions. All other mutants had < 10(-5) of wild-type activity. None of the purified mutants that exhibited catalytic activity had a significantly altered Km for paraoxon.

Crystallization and preliminary X-ray analysis of methionine aminopeptidase from the hyperthermophilic bacterium Pyrococcus furiosus.

Methionine aminopeptidase (MAP) from Pyrococcus furiosus (Pfu) has been crystallized in four different forms (A, B, C and D). Form A crystals belong to space group P2(1) with unit-cell dimensions a = 54.18, b = 85.72, c = 72.84 A, beta = 108.34 degrees. Forms B, C and D belong to space group P6(2(4)) with unit-cell dimensions a = 139.1, c = 63.7 A for form B, a = 198.6, c = 243.8 A for form C, and a = 111.0, c = 125.0 A for form D. Forms A and D diffract to 2.9 A, form B diffracts to 3.5 A, and form C crystals diffract to 4.5 A. Form A contains two molecules of MAP-Pfu per asymmetric unit. The binuclear metal center positions and a non-crystallographic twofold symmetry matrix has been determined for the form A crystals.

Overexpression and purification of human calcineurin alpha from Escherichia coli and assessment of catalytic functions of residues surrounding the binuclear metal center.

Calcineurin is an important signal-transducing enzyme in many cell types including T lymphocytes and is a common target for the immunosuppressants cyclosporin A and FK506. The crystal structures of both calcineurin [Griffith et al. (1995) Cell 82, 507-522; Kissinger et al. (1995) Nature 378, 641-644] and a related enzyme, protein phosphatase-1 [Goldberg et al. (1995) Nature 376, 745-753], revealed that this class of serine/threonine phosphatases contain in their putative active sites a binuclear metal center formed by an Asn, two Asp, and three His residues. In addition, one His and two Arg residues lie in close vicinity of the binuclear metal centers. The importance of the binuclear metal center and its surrounding residues in catalysis by calcineurin has not been investigated experimentally. Herein, we report an efficient bacterial expression and purification system for human calcineurin alpha. Using this system, a systematic alanine-scan mutagenesis on the residues surrounding the putative active site was performed. It was found that an intact binuclear metal center is essential for the catalytic activity of the enzyme. In addition, His151, Arg122, and Arg254 also exhibited either a loss or a dramatic decrease in catalytic activity upon mutation into alanines. Interestingly, the Arg254Ala mutant retained a small but significant amount of catalytic activity toward the small substrate p-nitrophenyl phosphate, but is completely inactive toward a phosphopeptide substrate, suggesting that this arginine may be involved in the binding of phosphoprotein substrates as well as in catalysis. As all the residues in the putative active site are conserved between different eukaryotic serine/threonine phosphatases, these results should apply to all members of this family of protein phosphatases.

Aspartate-407 in Rhodobacter sphaeroides cytochrome c oxidase is not required for proton pumping or manganese binding.

Several pathways for proton transport in cytochrome c oxidase have been proposed on the basis of mutational analysis and X-ray structure: at least one for moving "pumped" protons from the interior to exterior of the membrane and a separate route for transporting "substrate" protons from the interior to the binuclear metal center to combine with oxygen to make H2O. According to the crystal structures of cytochrome c oxidase, Asp407 (Rhodobacter sphaeroides numbering) is at the interface of subunit I and subunit II of the oxidase, in a negative patch proposed to be the proton exit site in a pumping pathway, as well as a possible ligand to Mg [Iwata et al. (1995) Nature 376, 660-669]. Three mutants at the Asp407 position of R. sphaeroides cytochrome oxidase, Asp407Ala, Asp407Asn, and Asp407Cys, have been purified and characterized. All showed electron transfer activity, and pH dependence of activity, similar to that of the wild type enzyme and no major structural changes, as evidenced by visible, EPR, and resonance Raman spectroscopy. When reconstituted into artificial vesicles, the purified mutants pumped protons with normal efficiency and responded to the membrane pH and electrical gradients in a manner similar to that of wild type. Furthermore, the EPR spectra and Mn quantitation analysis of mutants grown in high Mn indicated no significant alteration in the Mn/Mg site. These results suggest that Asp407 does not play a critical role in proton translocation or in Mn/Mg binding.

Perturbations to the active site of phosphotriesterase.

Phosphotriesterase catalyzes the hydrolysis of organophosphate nerve agents. Four amino acid residues, located within the active site pocket, were mutated in an effort to ascertain the roles that these groups play in the structure and function of this enzyme. Tryptophan-131 is located at the entrance to the binuclear metal center, and the indole ring is positioned to suggest that it could provide a hydrophobic site for interaction of the aromatic leaving group with optimized substrates. The W131F mutant displays catalytic constants for the hydrolysis of paraoxon that are essentially the same as those of the wild type enzyme. However, the Km value for the W131A mutant is elevated by a factor of 6, consistent with a role for this residue in substrate binding. Aspartate-253 is hydrogen bonded to His-230 which, in turn, is directly ligated to the more solvent-exposed metal ion. The D253N mutant possesses catalytic constants that are virtually the same as those of the wild type enzyme, while the D253A mutant is reduced in activity by 500-fold. These results are consistent with a model where this residue is required to orientate the imidazole side chain of His-230 for proper interaction with the binuclear metal center. Aspartate-301 is a primary ligand to the more buried metal ion. Mutation of this residue to histidine, asparagine, alanine, and cysteine reduces the catalytic activity by factors of 2.6 x 10(4), 2.7 x 10(3), 5.6 x 10(2), and 1.5 x 10(2), respectively. These results indicate that alterations to the direct metal ligands, even with residues that can strongly coordinate divalent cations, cause a severe disruption to the proper functioning of the active site. In the wild type enzyme, the side chain of Lys-169 is carbamylated and also acts as a bridge between the two divalent cations. Significant losses in catalytic activity are obtained upon mutation of this residue to either alanine, glutamate, arginine, or methionine. The loss in activity can partially be restored upon inclusion in the assay mixture of short-chain carboxylic acids. A 25-fold enhancement in k(cat) is observed for the K169A mutant in the presence of 100 mM propionic acid.

Crystal structure of the protein serine/threonine phosphatase 2C at 2.0 A resolution.

Protein phosphatase 2C (PP2C) is a Mn2+- or Mg2+-dependent protein Ser/Thr phosphatase that is essential for regulating cellular stress responses in eukaryotes. The crystal structure of human PP2C reveals a novel protein fold with a catalytic domain composed of a central beta-sandwich that binds two manganese ions, which is surrounded by alpha-helices. Mn2+-bound water molecules at the binuclear metal centre coordinate the phosphate group of the substrate and provide a nucleophile and general acid in the dephosphorylation reaction. Our model presents a framework for understanding not only the classical Mn2+/Mg2+-dependent protein phosphatases but also the sequence-related domains of mitochondrial pyruvate dehydrogenase phosphatase, the Bacillus subtilus phosphatase SpoIIE and a 300-residue domain within yeast adenyl cyclase. The protein architecture and deduced catalytic mechanism are strikingly similar to the PP1, PP2A, PP2B family of protein Ser/Thr phosphatases, with which PP2C shares no sequence similarity, suggestive of convergent evolution of protein Ser/Thr phosphatases.

Isolation, characterisation and expression of the bacterioferritin gene of Rhodobacter capsulatus

The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene ( bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18 174 Da. The molecular mass of the purified protein was estimated to be 18 176.06 ± 0.80 Da by electrospray mass spectrometry. The bfr gene was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those which provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conserved in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.

Isolation, characterisation and expression of the bacterioferritin gene ofRhodobacter capsulatus

The nucleotide sequence of the Rhodobacter capsulatus bacterioferritin gene (bfr) was determined and found to encode a protein of 161 amino acids with a predicted molecular mass of 18,174 Da. The molecular mass of the purified protein was estimated to be 18,176. +/ 0.80 Da by electrospray mass spectrometry. The bfr was introduced into an expression vector, and bacterioferritin was produced to a high level in Escherichia coli. The amino acids which are involved in haem ligation, and those provide ligands in the binuclear metal centre in bacterioferritin from E. coli are conversed in the R. capsulatus protein. The sequences of bacterioferritins, ferritin-like proteins, and proteins similar to Dps of E. coli are compared, and membership of the bacterioferritin family re-evaluated.

Metal-substrate interactions facilitate the catalytic activity of the bacterial phosphotriesterase.

The bacterial phosphotriesterase from Pseudomonas diminuta is a zinc metalloenzyme which catalyzes the hydrolysis of a variety of organophosphorus nerve agents with high efficiency. The active site of the enzyme consists of a coupled binuclear metal center embedded within a cluster of histidine residues. Potential protein-substrate interactions at the active site were probed by a systematic variation of metal identity, leaving group potential, phosphate host, and amino acid replacement. In order to determine the roles of these metal ions in binding and catalysis, the microscopic rate constants and kinetic parameters were obtained with various divalent cations. The divalent cations that were utilized in this investigation consisted of Co2+, Ni2+, Cd2+, Zn2+, Mn2+, and the mixed-metal Zn2+/Cd2+ hybrid. The leaving group potential and phosphate host were varied by altering the pKa of the departing substituted phenol or thiophenol in either a diethyl phosphate or a diethyl thiophosphate substrate. The Brønsted plots for the nonenzymatic hydroxide catalyzed hydrolysis of these substrates showed a linear dependence between the pseudo-first-order rate constant and the pKa of the leaving group. Enzymatic activities of the wild-type enzyme with these same substrates varied by over 7 orders of magnitude over the entire experimental pKa range (4.1-10.3), and the corresponding Brønsted plots were nonlinear. Those substrates with leaving groups with high pKa values were limited by the rate of bond cleavage while those substrates having leaving groups with low pKa values were limited by a conformational change or binding event. Thiophosphate substrates having leaving groups with high pKa values were better substrates than the corresponding phosphate analogues. These results are consistent with the direct coordination of one or both metal ions with the phosphoryl sulfur or oxygen atom of the substrate. A large dependence of the rate on the leaving group rules out the possibility of protonation of the leaving group or electrostatic interaction of the leaving group oxygen (or sulfur) with a metal ion or cationic group at the active site. The large differences in the size of the beta lg over the range of metal ions utilized by the enzyme indicate that the metal ions polarize the phosphoryl group and alter the structure of the transition state. The values of V/K(m) for the enzyme-catalyzed hydrolysis for a series of substituted thiophenol analogues were 10(2)-10(3)-fold smaller than those obtained for the hydrolysis of the corresponding phenolic substrates, suggesting that the bulkier sulfur substituent in the leaving group may induce conformational restrictions at the active site. With the zinc-substituted H201N mutant enzyme, there was a large decrease in the rate of phosphotriester hydrolysis but essentially no change in the rate of thiophosphotriester hydrolysis relative to the values observed for the zinc-substituted wild-type enzyme. These results suggest that a direct perturbation in the ligand structure of the binuclear metal center induces alterations in the mechanism of substrate hydrolysis.