RESUMO
Interpreting changes in patient genomes, understanding how viruses evolve and engineering novel protein function all depend on accurately predicting the functional outcomes that arise from amino acid substitutions. To that end, the development of first-generation prediction algorithms was guided by historic experimental datasets. However, these datasets were heavily biased toward substitutions at positions that have not changed much throughout evolution (i.e. conserved). Although newer datasets include substitutions at positions that span a range of evolutionary conservation scores, these data are largely derived from assays that agglomerate multiple aspects of function. To facilitate predictions from the foundational chemical properties of proteins, large substitution databases with biochemical characterizations of function are needed. We report here a database derived from mutational, biochemical, bioinformatic, structural, pathological and computational studies of a highly studied protein family-pyruvate kinase (PYK). A centerpiece of this database is the biochemical characterization-including quantitative evaluation of allosteric regulation-of the changes that accompany substitutions at positions that sample the full conservation range observed in the PYK family. We have used these data to facilitate critical advances in the foundational studies of allosteric regulation and protein evolution and as rigorous benchmarks for testing protein predictions. We trust that the collected dataset will be useful for the broader scientific community in the further development of prediction algorithms. Database URL https://github.com/djparente/PYK-DB.
Assuntos
Isoenzimas , Piruvato Quinase , Humanos , Piruvato Quinase/genética , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Isoenzimas/metabolismo , Ligantes , Proteínas/química , Regulação Alostérica , Biologia ComputacionalRESUMO
The allosteric coupling constant in K-type allosteric systems is defined as a ratio of the binding of substrate in the absence of effector to the binding of the substrate in the presence of a saturating concentration of effector. As a result, the coupling constant is itself an equilibrium value comprised of a ΔH and a TΔS component. In the scenario in which TΔS completely compensates ΔH, no allosteric influence of effector binding on substrate affinity is observed. However, in this "silent coupling" scenario, the presence of effector causes a change in the ΔH associated with substrate binding. A suggestion has now been made that "silent modulators" are ideal drug leads because they can be modified to act as either allosteric activators or inhibitors. Any attempt to rationally design the effector to be an allosteric activator or inhibitor is likely to be benefitted by knowledge of the mechanism that gives rise to coupling. Hydrogen/deuterium exchange with mass spectrometry detection has now been used to identify regions of proteins that experience conformational and/or dynamic changes in the allosteric regulation. Here, we demonstrate the expected temperature dependence of the allosteric regulation of rabbit muscle pyruvate kinase by Ala to demonstrate that this effector reduces substrate (phosphoenolpyruvate) affinity at 35°C and at 10°C but is silent at intermediate temperatures. We then explore the use of hydrogen/deuterium exchange with mass spectrometry to evaluate the areas of the protein that are modified in the mechanism that gives rise to the silent coupling between Ala and phosphoenolpyruvate. Many of the peptide regions of the protein identified as changing in this silent system (Ala as the effector) were included in changes previously identified for allosteric inhibition by Phe.
Assuntos
Peptídeos , Proteínas , Regulação Alostérica , Animais , Entropia , Coelhos , TermodinâmicaRESUMO
13C Metabolic Flux Analysis (13C-MFA) involves the quantification of isotopic enrichment in cellular metabolites and fitting the resultant data to the metabolic network model of the organism. Coverage and resolution of the resultant flux map depends on the total number of metabolites and fragments in which 13C enrichment can be quantified accurately. Experimental techniques for tracking 13C enrichment are evolving rapidly and large volumes of data are now routinely generated through the use of Liquid Chromatography coupled with High-Resolution Mass Spectrometry (HR-LC/MS). Therefore, the current manuscript is focused on the challenges in high-throughput analyses of such large datasets. Current 13C-MFA studies often have to rely on the targeted quantification of a small subset of metabolites, thereby leaving a large fraction of the data unexplored. A number of public domain software tools have been reported in recent years for the untargeted quantitation of isotopic enrichment. However, the suitability of their application across diverse datasets has not been investigated. Here, we test the software tools X13CMS, DynaMet, geoRge, and HiResTEC with three diverse datasets. The tools provided a global, untargeted view of 13C enrichment in metabolites in all three datasets and a much-needed automation in data analysis. Some inconsistencies were observed in results obtained from the different tools, which could be partially ascribed to the lack of baseline separation and potential mass conflicts. After removing the false positives manually, isotopic enrichment could be quantified reliably in a large repertoire of metabolites. Of the software tools explored, geoRge and HiResTEC consistently performed well for the untargeted analysis of all datasets tested.
RESUMO
13C Metabolic Flux Analysis (13C-MFA) is a powerful tool for quantification of carbon flux distribution in metabolic pathways. However, the requirement to obtain accurate labeling patterns, especially for compounds with low abundance, poses a challenge. Chromatographic separation and high sensitivity of the modern mass spectrometers (MS) alleviate this problem to a certain extent. However, the presence of derivatives such as in-source fragments, multimer ion adducts, and multiply charged ions result in reduced intensity of the molecular ion. While multimer ion adducts have been reported in the field of metabolomics, their presence is considered undesirable in quantitative studies. Here, we demonstrate a novel application of dimer ion adducts in calculating the mass isotopologue distribution (MIDs) of the corresponding monomer ions for public domain and in-house generated datasets comprising of 13C-labeling time-course experiments. Out of the 100 standard compounds analyzed, we could detect multimer ion adducts in 24 of the intermediate metabolites. Further, a subset of these multimer ions were detected in all the biological samples analyzed. Majority of these ion adducts were either not detected in the original study or labeled as a putative features. Regression analysis was performed to estimate the monomer MIDs from those of the dimer. This resulted in accurate estimation regardless of the biological system, chromatographic method, the MS hardware, or the relative abundance of the dimer ion. We argue that this analysis may be useful in cases where satisfactory data cannot be extracted from the chromatographic peaks of the monomer ions.
Assuntos
Isótopos de Carbono/análise , Análise do Fluxo Metabólico/métodos , Marcação por Isótopo/métodos , Espectrometria de Massas/métodos , Redes e Vias Metabólicas , Metabolômica/métodosRESUMO
The environmental considerations attributing to the escalation of carbon dioxide emissions have raised alarmingly. Consequently, the concept of sequestration and biological conversion of CO2 by photosynthetic microorganisms is gaining enormous recognition. In this study, in an attempt to discern the synergistic CO2 tolerance mechanisms, metabolic responses to increasing CO2 concentrations were determined for Synechococcus elongatus PCC 11801, a fast-growing, novel freshwater strain, using quantitative proteomics. The protein expression data revealed that the organism responded to elevated CO2 by not only regulating the cellular transporters involved in carbon-nitrogen uptake and assimilation but also by inducing photosynthesis, carbon fixation and glycolysis. Several components of photosynthetic machinery like photosystem reaction centers, phycobilisomes, cytochromes, etc. showed a marked up-regulation with a concomitant downshift in proteins involved in photoprotection and redox maintenance. Additionally, enzymes belonging to the TCA cycle and oxidative pentose phosphate pathway exhibited a decline in their expression, further highlighting that the demand for reduced cofactors was fulfilled primarily through photosynthesis. The present study brings the first-ever comprehensive assessment of intricate molecular changes in this novel strain while shifting from carbon-limited to carbon-sufficient conditions and may pave the path for future host and pathway engineering for production of sustainable fuels through efficient CO2 capture.
Assuntos
Proteínas de Bactérias/metabolismo , Dióxido de Carbono , Synechococcus/crescimento & desenvolvimento , Synechococcus/metabolismo , Dióxido de Carbono/farmacologia , Biologia Computacional , Proteoma/metabolismo , Proteômica/métodos , Reprodutibilidade dos Testes , Estresse Fisiológico , Synechococcus/efeitos dos fármacosRESUMO
A key requirement for 13C Metabolic flux analysis (13C-MFA), a widely used technique to estimate intracellular metabolic fluxes, is an efficient method for the extraction of intermediate metabolites for analysis via liquid chromatography mass spectrometry (LC/MS). The 13C isotopic labeling results in further distribution of an already sparse pool of intermediate metabolites into isotopologues, each appearing as a separate chromatographic feature. We examined some of the reported solvent systems for the extraction of polar intracellular metabolites from three strains of cyanobacteria of the genus Synechococcus, viz., Synechococcus sp. PCC 7002, Synechococcus elongatus PCC 7942, and a newly isolated Synechococcus elongatus PCC 11801 (manuscript under review). High resolution-LC/MS was used to assess the relative abundance of the extracted metabolites. The different solvent systems used for extraction led to statistically significant changes in the extraction efficiency for a large number of metabolites. While a few hundred m/z features or potential metabolites were detected with different solvent systems, the abundance of over a quarter of all metabolites varied significantly from one solvent system to another. Further, the extraction methods were evaluated for a targeted set of metabolites that are important in 13C-MFA studies of photosynthetic organisms. While for the strain PCC 7002, the reported method using methanol-chloroform-water system gave satisfactory results, a mild base in the form of NH4OH had to be used in place of water to achieve adequate levels of extraction for PCC 7942 and PCC 11801. While minor changes in extraction solvent resulted in dramatic changes in the extraction efficiency of a number of compounds, certain metabolites such as amino acids and organic acids were adequately extracted in all the solvent systems tested. Overall, we present a new improved method for extraction using a methanol-chloroform-NH4OH system. Our method improves the extraction of polar compounds such as sugar phosphates, bisphosphates, that are central to 13C-MFA studies.
Assuntos
Análise do Fluxo Metabólico/métodos , Metabolômica/métodos , Solventes/análise , Synechococcus/química , Hidróxido de Amônia/análise , Isótopos de Carbono/química , Clorofórmio/análise , Cromatografia Líquida/métodos , Metanol/análise , Espectrometria de Massas em Tandem/métodosRESUMO
Accurate quantification of mass isotopologue distribution (MID) of metabolites is a prerequisite for 13C-metabolic flux analysis. Currently used mass spectrometric (MS) techniques based on multiple reaction monitoring (MRM) place limitations on the number of MIDs that can be analyzed in a single run. Moreover, the deconvolution step results in amplification of error. Here, we demonstrate that SWATH MS/MS, a data independent acquisition (DIA) technique allows quantification of a large number of precursor and product MIDs in a single run. SWATH sequentially fragments all precursor ions in stacked mass isolation windows. Co-fragmentation of all precursor isotopologues in a single SWATH window yields higher sensitivity enabling quantification of MIDs of fragments with low abundance and lower systematic and random errors. We quantify the MIDs of 53 precursor and product ions corresponding to 19 intracellular metabolites from a dynamic 13C-labeling of a model cyanobacterium, Synechococcus sp. PCC 7002. The use of product MIDs resulted in an improved precision of many measured fluxes compared to when only precursor MIDs were used for flux analysis. The approach is truly untargeted and allows additional metabolites to be quantified from the same data.
Assuntos
Isótopos de Carbono/análise , Análise do Fluxo Metabólico/métodos , Synechococcus/metabolismo , Espectrometria de Massas em Tandem/métodos , Isótopos de Carbono/metabolismo , Synechococcus/química , Fluxo de TrabalhoRESUMO
Cyanobacteria, which constitute a quantitatively dominant phylum, have attracted attention in biofuel applications due to favorable physiological characteristics, high photosynthetic efficiency and amenability to genetic manipulations. However, quantitative aspects of cyanobacterial metabolism have received limited attention. In the present study, we have performed isotopically non-stationary 13 C metabolic flux analysis (INST-13 C-MFA) to analyze rerouting of carbon in a glycogen synthase deficient mutant strain (glgA-I glgA-II) of the model cyanobacterium Synechococcus sp. PCC 7002. During balanced photoautotrophic growth, 10-20% of the fixed carbon is stored in the form of glycogen via a pathway that is conserved across the cyanobacterial phylum. Our results show that deletion of glycogen synthase gene orchestrates cascading effects on carbon distribution in various parts of the metabolic network. Carbon that was originally destined to be incorporated into glycogen gets partially diverted toward alternate storage molecules such as glucosylglycerol and sucrose. The rest is partitioned within the metabolic network, primarily via glycolysis and tricarboxylic acid cycle. A lowered flux toward carbohydrate synthesis and an altered distribution at the glucose-1-phosphate node indicate flexibility in the network. Further, reversibility of glycogen biosynthesis reactions points toward the presence of futile cycles. Similar redistribution of carbon was also predicted by Flux Balance Analysis. The results are significant to metabolic engineering efforts with cyanobacteria where fixed carbon needs to be re-routed to products of interest. Biotechnol. Bioeng. 2017;114: 2298-2308. © 2017 Wiley Periodicals, Inc.
Assuntos
Carbono/metabolismo , Cianobactérias/fisiologia , Glicogênio Sintase/genética , Glicogênio/genética , Glicogênio/metabolismo , Análise do Fluxo Metabólico/métodos , Redes e Vias Metabólicas/fisiologia , Isótopos de Carbono/farmacologia , Simulação por Computador , Cianobactérias/classificação , Cianobactérias/efeitos da radiação , Luz , Taxa de Depuração Metabólica/efeitos da radiação , Redes e Vias Metabólicas/efeitos da radiação , Modelos Biológicos , Mutação/genética , Fotossíntese/fisiologia , Fotossíntese/efeitos da radiaçãoRESUMO
Flux Balance Analysis was performed with the Genome Scale Metabolic Model of a fast growing cyanobacterium Synechococcus sp. PCC 7002 to gain insights that would help in engineering the organism as a production host. Gene essentiality and synthetic lethality analysis revealed a reduced metabolic robustness under genetic perturbation compared to the heterotrophic bacteria Escherichia coli. Under glycerol heterotrophy the reducing equivalents were generated from tricarboxylic acid cycle rather than the oxidative pentose phosphate pathway. During mixotrophic growth in glycerol the photosynthetic electron transport chain was predominantly used for ATP synthesis with a photosystem I/photosystem II flux ratio higher than that observed under autotrophy. An exhaustive analysis of all possible double reaction knock outs was performed to reroute fixed carbon towards ethanol and butanol production. It was predicted that only â¼10% of fixed carbon could be diverted for ethanol and butanol production.
Assuntos
Biocombustíveis , Genes Bacterianos , Modelos Biológicos , Synechococcus/genética , Synechococcus/metabolismo , Processos Autotróficos , Butanóis/metabolismo , Ciclo do Carbono , Ciclo do Ácido Cítrico , Etanol/metabolismo , Genoma Bacteriano , Glicerol/metabolismo , Processos Heterotróficos , Mutação , Via de Pentose Fosfato , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Reprodutibilidade dos TestesRESUMO
Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e., a negligible reduction in the affinity of rM1-PYK for the substrate, phosphoenolpyruvate. This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e., those changes common upon the addition of alanine vs phenylalanine) from changes associated with allosteric regulation (i.e., those elicited by the addition of phenylalanine binding, but not alanine binding). High-quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique includes residues previously identified by mutagenesis to have roles in allosteric regulation by phenylalanine.
Assuntos
Músculos/enzimologia , Fenilalanina/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Regulação Alostérica , Sítio Alostérico , Animais , Espectrometria de Massas , Modelos Moleculares , Músculos/química , Músculos/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica , CoelhosRESUMO
During our efforts to characterize the regulatory properties of human liver pyruvate kinase (L-PYK), we have noted that the affinity of the protein for phosphoenolpyruvate (PEP) becomes reduced several days after cell lysis. A 1.8 Å crystallographic structure of L-PYK with the S12D mimic of phosphorylation indicates that Cys436 is oxidized, the first potential insight into explaining the effect of "aging". Interestingly, the oxidation is only to sulfenic acid despite the crystal growth time period of 2 weeks. Mutagenesis confirms that the side chain of residue 436 is energetically coupled to PEP binding. Mass spectrometry confirms that the oxidation is present in solution and is not an artifact caused by X-ray exposure. Exposure of the L-PYK mutations to H2O2 also confirms that PEP affinity is sensitive to the nature of the side chain at position 436. A 1.95 Å structure of the C436M mutant of L-PYK, the only mutation at position 436 that has been shown to strengthen PEP affinity, revealed that the methionine substitution results in the ordering of several N-terminal residues that have not been ordered in previous structures. This result allowed speculation that oxidation of Cys436 and phosphorylation of the N-terminus at Ser12 may function through a similar mechanism, namely the interruption of an activating interaction between the nonphosphorylated N-terminus with the nonoxidized main body of the protein. Mutant cycles were used to provide evidence that mutations of Cys436 are energetically synergistic with N-terminal modifications, a result that is consistent with phosphorylation of the N-terminus and oxidation of Cys436 functioning through mechanisms with common features. Alanine-scanning mutagenesis was used to confirm that the newly ordered N-terminal residues were important to the regulation of enzyme function by the N-terminus of the enzyme (i.e., not an artifact caused by the introduced methionine substitution) and to further define which residues in the N-terminus are energetically coupled to PEP affinity. Collectively, these studies indicate energetic coupling (and potentially mechanistic similarities) between the oxidation of Cys436 and phosphorylation of Ser12 in the N-terminus of L-PYK.
Assuntos
Cisteína/metabolismo , Fígado/enzimologia , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Alquilação , Biocatálise , Cristalografia por Raios X , Cisteína/química , Humanos , Cinética , Ligantes , Conformação Molecular , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Acoplamento Oxidativo , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fosfoenolpiruvato/química , Fosfoenolpiruvato/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Piruvato Quinase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
An advantage of studying allosteric regulation over covalent modification is that allostery allows the experimentalist to vary the concentration of effector, thereby allowing independent quantification of effector binding and allosteric coupling. In turn, this capacity allows the use of effector analogues to determine which regions of the effector contribute to effector binding and which contribute to allosteric regulation. Like many other proteins, human liver pyruvate kinase (hL-PYK) is regulated by phosphorylation. The phosphorylation of hL-PYK occurs on Ser12 of the N-terminus. Phosphorylation appears to interrupt an interaction (distant from the active site) between the N-terminus and the main body of the protein. Since this interaction increases the affinity of hL-PYK for the substrate (phosphoenolpyruvate, PEP), phosphorylation-dependent interruption of the N-terminus/main-body interaction results in an antagonism of PEP binding. Due to the advantages of studying an allosteric system, we detail a protocol to express and purify N-terminal peptides of hL-PYK using a SUMO-fusion system. We further demonstrate that these peptides act as allosteric regulators that modulate the affinity of hL-PYK for PEP.
Assuntos
Fígado/enzimologia , Peptídeos/farmacologia , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/genética , Humanos , Peptídeos/química , Fosforilação/genética , Fosforilação/fisiologia , Conformação ProteicaRESUMO
Hydrogen/deuterium exchange in combination with mass spectrometry (H/D MS) is a sensitive technique for detection of changes in protein conformation and dynamics. However, wide application of H/D MS has been hindered, in part, by the lack of computational tools necessary for efficient analysis of the large data sets associated with this technique. We report a novel web-based application for automatic analysis of H/D MS experimental data. This application relies on the high resolution of mass spectrometers to extract all isotopic envelopes before correlating these envelopes with individual peptides. Although a fully automatic analysis is possible, a variety of graphical tools are included to aid in the verification of correlations and rankings of the isotopic peptide envelopes. As a demonstration, the rate constants for H/D exchange of peptides from rabbit muscle pyruvate kinase are mapped onto the structure of this protein.
Assuntos
Biologia Computacional/métodos , Medição da Troca de Deutério/métodos , Espectrometria de Massas/métodos , Peptídeos/química , Software , Animais , Bases de Dados de Proteínas , Internet , Modelos Moleculares , Peptídeos/metabolismo , Piruvato Quinase/química , Piruvato Quinase/metabolismo , Coelhos , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Nature has illustrated through numerous examples that protein dimerization has structural and functional advantages. We previously reported the design and characterization of an engineered "metallohomeodomain" protein (C2) based on a chimera of the EF-hand Ca-binding motif and the helix-turn-helix motif of homeodomains (Lim and Franklin in Protein Sci. 15:2159-2165, 2004). This small metalloprotein binds the hard metal ions Ca(II) and Ln(III) and interacts with DNA with modest sequence preference and affinity, yet exhibits only residual DNA cleavage activity. Here we have achieved substantial improvement in function by constructing a covalent dimer of this C2 module (F2) to create a larger multidomain protein. As assayed via fluorescence spectroscopy, this F2 protein binds Ca(II) more avidly (25-fold) than C2 on a per-domain basis; in gel shift selection experiments, metallated F2 exhibits a specificity toward 5'-TAATTA-3' sequences. Finally, Ca(2)F2 cleaves plasmid DNA and generates a linear product in a Ca(II)-dependent way, unlike the CaC2 monomer. To the best of our knowledge this activation of Ca(II) in the context of an EF-hand binding motif is unique and represents a significant step forward in the design of artificial metallonucleases by utilizing biologically significant metal ions.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Metaloproteínas/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Sequência de Bases , DNA/química , DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Dimerização , Motivos EF Hand , Eletroforese em Gel de Poliacrilamida , Metaloproteínas/química , Metaloproteínas/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
There is currently a renewed focus aimed at understanding allosteric mechanisms at atomic resolution. This current interest seeks to understand how both changes in protein conformations and changes in protein dynamics contribute to relaying an allosteric signal between two ligand binding sites on a protein (e.g., active and allosteric sites). Both nuclear magnetic resonance (NMR), by monitoring protein dynamics directly, and hydrogen/deuterium exchange, by monitoring solvent accessibility of backbone amides, offer insights into protein dynamics. Unfortunately, many allosteric proteins exceed the size limitations of standard NMR techniques. Although hydrogen/deuterium exchange as detected by mass spectrometry (H/DX-MS) offers an alternative evaluation method, any application of hydrogen/deuterium exchange requires that the property being measured functions in both H(2)O and D(2)O. Due to the promising future H/DX-MS has in the evaluation of allosteric mechanisms in large proteins, we demonstrate an evaluation of allosteric regulation in D(2)O. Exemplified using phenylalanine inhibition of rabbit muscle pyruvate kinase, we find that binding of the inhibitor is greatly reduced in D(2)O, but the effector continues to elicit an allosteric response.
Assuntos
Medição da Troca de Deutério , Deutério/análise , Regulação Alostérica , Animais , Sítios de Ligação , Hidrogênio/análise , Espectroscopia de Ressonância Magnética , Modelos Biológicos , Coelhos , TermodinâmicaRESUMO
To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation. Here, we test global kinetic analysis as an approach to this problem. Taking advantage of the various binding and cleavage constants established for PvuII endonuclease, we apply cleavage data obtained under a range of Mg(II) and Ca(II) concentrations to a number of kinetic models which specify A and B sites for both metal ions and various active species. The data are best fit and simulated with models which feature Ca(II) being held more strongly in the B (or secondary) site. This mixed metal enzyme species is the only one which forms appreciably and exhibits a cleavage rate constant similar to that observed when there is only one Mg(II) per active site (approximately 0.01 s(-1)). Thus, in the case of PvuII endonuclease, Ca(II) does not stimulate cleavage. However, a simulated increase in activity at moderate Ca(II) concentrations can be rationalized with a cleavage rate constant for the mixed species similar to that when two Mg(II) ions are present in the active site. This provides an important insight into the underlying basis for the Ca(II)-stimulated activity observed for some metallonucleases that is not accessible by any other means.