Removal of Metal from Carboxypeptidase A Proceeds via a Split Pathway: Implications for the General Mechanisms of Metalloenzyme Inactivation by Chelating Agents.

The chelation of protein-bound metal ions is typically thought to follow either a dissociative (D) or an associative (A) path. While the former mechanism involves the spontaneous dissociation of the metal from the protein prior to chelation, the latter route is characterized by the formation of an intermediate protein-metal-chelator ternary complex. Using the prototypical zinc protease carboxypeptidase A (CPA) and a variety of charged and neutral chelating agents, we demonstrate that inactivation of the enzyme (and likely other metalloproteins) proceeds through a split pathway with contributions from both D- and A-type mechanisms. In the case of charged chelators such as ethylenediaminetetraacetic acid (EDTA), the proportions of both paths could be tuned over a wide range through variation of the chelator concentration and the ionic strength, I (from ∼95% A type at low I values to ∼5% at high I values). By measuring the EDTA concentration and time dependence of CPA inactivation and fitting the obtained kinetic data to a modified time-dependent inhibition model, we obtained the rate constants for the A and D paths (kinact and koff, respectively) and the inhibition constant (KI) for the formation of the CPA-Zn2+-EDTA ternary complex, indicating that the decreased contribution of the A-type mechanism at high ionic strengths originates from a large (40-fold; at I = 0.5 M) increase in KI. This observation might be related to a triarginine motif in CPA that electrostatically steers negatively charged substrates into the active site and may therefore also guide carboxylate-bearing chelators toward the Zn2+ ion.

An Excel fitting routine for correcting protein absorption spectra for scatter.

Protein concentrations are often determined in a non-destructive manner by measuring the absorbance at 280 nm. However, light scattering in protein samples can complicate such assessment. We here describe a simple Excel Solver-based fitting routine to correct full protein UV absorption spectra for both Rayleigh and Mie scattering. Using samples displaying various degrees of natural and artificially induced scattering, we show that our multi-wavelength fitting method is not only capable of aiding in the determination of protein concentrations but can also be employed in the spectral analysis of protein structural changes that are accompanied by alterations in scatter intensity.

Effect of pH and denaturants on the fold and metal status of anthrax lethal factor.

Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn2+ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn2+ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.

Specificity-directed design of a FRET-quenched heptapeptide for assaying thermolysin-like proteases.

Thermolysin (TL) is an industrially important zinc endopeptidase, and the prototype of the M4 family of metallopeptidases. The catalytic function of TL and its relatives is typically assessed using chromogenic or more sensitive fluorescent peptides, with the latter substrates relying on Förster resonance energy transfer (FRET). Here, we demonstrate that a FRET-quenched heptapeptide designed on the basis of the enzyme's substrate specificity (Dabcyl-FKFLGKE-EDANS) is efficiently cleaved by TL and dispase (a TL-like protease) in between the Phe3 and Leu4 residues. The specificity constants (determined at pH 7.4 and 25 °C) for TL and dispase (3.6 × 106 M-1 s-1 and 4.6 × 106 M-1 s-1, respectively) were found to be amongst the highest documented for any TL substrate. Maximal peptide cleavage rates were achieved at pH 6.5 and a temperature of 65 °C. In view of the sensitivity of the assay, concentrations as low as 10 pM TL could be detected. Furthermore, the rate of hydrolysis of Dabcyl-FKFLGKE-EDANS was slow or immeasurable with some other unrelated metallo-, serine- and cysteine proteases, suggesting that the peptide has the potential to serve as a selective substrate for TL and TL-like proteases.

Structural and Kinetic Studies of the Potent Inhibition of Metallo-ß-lactamases by 6-Phosphonomethylpyridine-2-carboxylates.

There are currently no clinically available inhibitors of metallo-ß-lactamases (MBLs), enzymes that hydrolyze ß-lactam antibiotics and confer resistance to Gram-negative bacteria. Here we present 6-phosphonomethylpyridine-2-carboxylates (PMPCs) as potent inhibitors of subclass B1 (IMP-1, VIM-2, and NDM-1) and B3 (L1) MBLs. Inhibition followed a competitive, slow-binding model without an isomerization step (IC50 values of 0.3-7.2 µM; Ki values of 0.03-1.5 µM). Minimum inhibitory concentration assays demonstrated potentiation of ß-lactam (Meropenem) activity against MBL-producing bacteria, including clinical isolates, at concentrations at which eukaryotic cells remain viable. Crystal structures revealed unprecedented modes of binding of inhibitor to B1 (IMP-1) and B3 (L1) MBLs. In IMP-1, binding does not replace the nucleophilic hydroxide, and the PMPC carboxylate and pyridine nitrogen interact closely (2.3 and 2.7 Å, respectively) with the Zn2 ion of the binuclear metal site. The phosphonate group makes limited interactions but is 2.6 Å from the nucleophilic hydroxide. Furthermore, the presence of a water molecule interacting with the PMPC phosphonate and pyridine N-C2 π-bond, as well as the nucleophilic hydroxide, suggests that the PMPC binds to the MBL active site as its hydrate. Binding is markedly different in L1, with the phosphonate displacing both Zn2, forming a monozinc enzyme, and the nucleophilic hydroxide, while also making multiple interactions with the protein main chain and Zn1. The carboxylate and pyridine nitrogen interact with Ser221 and -223, respectively (3 Å distance). The potency, low toxicity, cellular activity, and amenability to further modification of PMPCs indicate these and similar phosphonate compounds can be further considered for future MBL inhibitor development.

Arginine-containing peptides as potent inhibitors of VIM-2 metallo-ß-lactamase.

BACKGROUND: Metallo-ß-lactamases (MBLs) play an important role in the emergence of microbial resistance to ß-lactam antibiotics, and are hence considered targets for the design of novel therapeutics. We here report on the inhibitory effect of peptides containing multiple arginine residues on VIM-2, a clinically important MBL from Pseudomonas aeruginosa. METHODS: Enzyme kinetic assays in combination with fluorescence spectroscopy and stopped-flow UV-Vis spectrophotometry were utilized to explore the structure-activity relationship of peptides as inhibitors of VIM-2. RESULTS: Our studies show that the inhibitory potency of the investigated peptides was mainly dependent on the number of arginine residues in the center of the peptide sequence, and on the composition of the N-terminus. The most potent inhibitors were found to curtail enzyme function in the mid-to-low nanomolar range. Salts generally reduced peptide-mediated inhibition. Analysis of the mode of inhibition suggests the peptides to act as mixed-type inhibitors with a higher affinity for the enzyme-substrate complex. Stopped-flow UV-Vis and fluorescence studies revealed the peptides to induce rapid protein aggregation, a phenomenon strongly correlated to the peptides' inhibitory potency. Inhibition of IMP-1 (another subclass B1 MBL) by the peptides was found to be much weaker than that observed with VIM-2, a finding which might be related to subtle molecular differences in the protein surfaces. CONCLUSION: The reported data indicate that arginine-containing peptides can serve as potent, aggregation-inducing inhibitors of VIM-2, and potentially of other MBLs. GENERAL SIGNIFICANCE: Arginine-containing peptides can be considered as a novel type of potent MBL inhibitors.

Assay for drug discovery: Synthesis and testing of nitrocefin analogues for use as ß-lactamase substrates.

We report on the synthesis of three nitrocefin analogues and their evaluation as substrates for the detection of ß-lactamase activity. These compounds are hydrolyzed by all four Ambler classes of ß-lactamases. Kinetic parameters were determined with eight different ß-lactamases, including VIM-2, NDM-1, KPC-2, and SPM-1. The compounds do not inhibit the growth of clinically important antibiotic-resistant gram-negative bacteria in vitro. These chromogenic compounds have a distinct absorbance spectrum and turn purple when hydrolyzed by ß-lactamases. One of these compounds, UW154, is easier to synthesize from commercial starting materials than nitrocefin and should be significantly less expensive to produce.

Preparation and characterization of cobalt-substituted anthrax lethal factor.

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of mitogen-activated protein kinase kinases near their N-termini. The current report concerns the preparation of cobalt-substituted LF (CoLF) and its characterization by electronic spectroscopy. Two strategies to produce CoLF were explored, including (i) a bio-assimilation approach involving the cultivation of LF-expressing Bacillus megaterium cells in the presence of CoCl(2), and (ii) direct exchange by treatment of zinc-LF with CoCl(2). Independent of the method employed, the protein was found to contain one Co(2+) per LF molecule, and was shown to be twice as active as its native zinc counterpart. The electronic spectrum of CoLF suggests the Co(2+) ion to be five-coordinate, an observation similar to that reported for other Co(2+)-substituted gluzincins, but distinct from that documented for the crystal structure of native LF. Furthermore, spectroscopic studies following the exposure of CoLF to thioglycolic acid (TGA) revealed a sequential mechanism of metal removal from LF, which likely involves the formation of an enzyme: Co(2+):TGA ternary complex prior to demetallation of the active site. CoLF reported herein constitutes the first spectroscopic probe of LF's active site, which may be utilized in future studies to gain further insight into the enzyme's mechanism and inhibitor interactions.

Alkaline earth metals are not required for the restoration of the apoform of anthrax lethal factor to its holoenzyme state.

Anthrax lethal factor (LF) is a zinc-dependent metalloendopeptidase previously shown to require calcium and magnesium for the restoration of its catalytic function upon exposure of the apoprotein (apoLF) to Zn(2+). Since concrete Ca(2+)/Mg(2+) binding sites have not been identified in LF, the effects of alkaline earth metals on the enzymatic function of holoLF (ZnLF) and on the reconstitution of apoLF were reinvestigated. The current study reveals alkaline earth metals to be inhibitory at concentrations higher than 1mM. A combination of activity/inhibition assays and Tb(3+) luminescence spectroscopy was employed to unequivocally establish the presence of at least one inhibitory low-affinity Ca(2+)-site in LF. A comparative analysis of apoLF preparations obtained by dialysis and centrifugal filtration (following treatment of ZnLF with chelators) revealed the exposure of apoLF to low equimolar amounts of Zn(2+) to be sufficient for the full restoration of the protein's catalytic competence, a finding constistent with the picomolar dissociation constant of ZnLF determined in this study. The previously documented requirement of Ca(2+) and Mg(2+) in apoLF reconstitution may be explicable on the basis of contamination of dialyzed apoprotein preparations with residual chelator, a phenomenon not encountered with apoLF obtained by centrifugal filtration.

A spectrophotometric method for the determination of zinc, copper, and cobalt ions in metalloproteins using Zincon.

Zincon (2-carboxy-2'-hydroxy-5'-sulfoformazylbenzene) has long been known as an excellent colorimetric reagent for the detection of zinc and copper ions in aqueous solution. To extend the chelator's versatility to the quantification of metal ions in metalloproteins, the spectral properties of Zincon and its complexes with Zn(2+), Cu(2+), and Co(2+) were investigated in the presence of guanidine hydrochloride and urea, two common denaturants used to labilize metal ions in proteins. These studies revealed the detection of metals to be generally more sensitive with urea. In addition, pH profiles recorded for these metals indicated the optimal pH for complex formation and stability to be 9.0. As a consequence, an optimized method that allows the facile determination of Zn(2+), Cu(2+), and Co(2+) with detection limits in the high nanomolar range is presented. Furthermore, a simple two-step procedure for the quantification of both Zn(2+) and Cu(2+) within the same sample is described. Using the prototypical Cu(2+)/Zn(2+)-protein superoxide dismutase as an example, the effectiveness of this method of dual metal quantification in metalloproteins is demonstrated. Thus, the spectrophotometric determination of metal ions with Zincon can be exploited as a rapid and inexpensive means of assessing the metal contents of zinc-, copper-, cobalt-, and zinc/copper-containing proteins.

Chelator-facilitated chemical modification of IMP-1 metallo-beta-lactamase and its consequences on metal binding.

A method involving the reversible chemical modification of an active site, zinc-binding cysteine residue (Cys221) for the specific removal of one of the two zinc ions in the metallo-beta-lactamase IMP-1 was explored. Covalent modification of Cys221 by 5,5'-dithio-bis(2-nitrobenzoic acid) was greatly enhanced by the presence of dipicolinic acid, and subsequent removal of the modifying group was easily achieved by reduction of the disulfide bond. However, mass spectrometric analyses and an assessment of IMP-1's catalytic competence are consistent with the maintenance of the enzyme's binuclear status. The consequences arising from chemical modification of Cys221 are thus distinct from those reported for Cys-->Ala/Ser mutants of IMP-1 and other metallo-beta-lactamases, which are mononuclear.

A direct spectrophotometric method for the simultaneous determination of zinc and cobalt in metalloproteins using 4-(2-pyridylazo)resorcinol.

An assay involving the direct and simultaneous determination of low micromolar concentrations (1-10 microM) of both zinc and cobalt ions suitable for metal content analyses of metalloproteins is described. The procedure exploits differences in the visible absorption spectra of the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) resulting from its complexation to Zn2+ and/or Co2+ ions and is based on the fit of experimental spectra to a linear addition of Beer-Lambert law. The method eliminates the need for separating or masking one of the metal ions prior to their quantification and could prove to be particularly useful in studies on Co2+-substituted zinc proteins.

Binding of Cu, Co, and Cs to fluorescent components of natural organic matter (NOM) from three contrasting sites.

Aqueous natural organic matter (NOM) contains different types of functional groups (carboxylic, phenolic, sulfidic, etc.), and hence could change the speciation of metals in environmental systems. This work is a proof-of-concept study on the interaction of three metals (Cu, Co, and Cs) with NOM using fluorescence spectroscopy. The specific aim was to determine the conditional stability constants for these three metals with NOM optical components, obtained from the quenching of fluorescence signals. Three contrasting water types were sampled in Northern Ontario: a pristine source (Cross Rd.), an urban-impacted source (Junction Creek), and an industrially impacted creek (Copper Cliff creek). In this investigation, Cu2+ was used as a benchmark, whereas Co2+ and Cs+ analyses were novel applications of this technique. Humic-like (H-like; terrestrial and microbial), fulvic-like (F-like), and protein-like (P-like) fluorescence components were found in various proportions at the three sampling sites. For these samples, the fluorescence signals of the H-, F-, and P-like components were quenched upon additions of Cu2+. The computed conditional stability constants (as log Kc) ranged from 4.46 to 6.06. In contrast, Kc values with Co2+ were measurable only for the two H-like components of the pristine sample (log Kc 3.02-4.05). Cesium (Cs+) induced quenching only for the P-like component at the industrial-impacted site (log Kc 4.82-5.03). While this study corroborates earlier reports that Cu2+-NOM interactions can be measured by fluorescence, we are showing for the first time a direct chemical interaction of Co2+ and Cs+ with specific NOM components, as reported by fluorescence quenching.

An investigation of the pH dependence of copper-substituted anthrax lethal factor and its mechanistic implications.

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase involved in the cleavage of proteins critical to the maintenance of host signaling pathways during anthrax infections. Although zinc is typically regarded as the native metal ion in vivo, LF is highly tolerant to metal substitution, with its replacement by copper yielding an enzyme (CuLF) 4.5-fold more active than the native zinc protein (at pH 7). The current study demonstrates that by careful choice of the buffer, ionic strength, pH and substrate, the activity ratio of CuLF and native LF can be increased to >40-fold. Using a fluorogenic LF substrate, such optimized assay conditions can be exploited to detect LF concentrations as low as 2 pM. In contrast to the zinc form, CuLF was found to be inhibited by bromide and iodide ions, to be resistant to metal loss under acidic conditions, and to display a sharp pH dependence with significantly shifted alkaline limb towards more acidic conditions. The alkaline limb in the enzyme's pH profile is suggested to originate from changes in the protonation state of the metal-bound water molecule which serves as the nucleophile in the catalytic mechanism. Based on these observations and studies on other zinc proteases, a minimal mechanism for LF is proposed.

68Zn isotope exchange experiments reveal an unusual kinetic lability of the metal ions in the di-zinc form of IMP-1 metallo-beta-lactamase.

The apparently paradoxical behaviour of facile exchange (kinetic lability) of tightly bound (thermodynamic stability) zinc ions in the enzyme IMP-1 metallo-beta-lactamase with Zn-68 and cadmium ions, as indicated by in-torch vaporization inductively-coupled plasma mass spectrometry (ITV-ICP-MS) and electrospray-ionization mass spectrometry (ESI-MS), is consistent with the involvement of a third metal ion in promoting Lewis acid/base type exchange processes.

Highly dynamic metal exchange in anthrax lethal factor involves the occupation of an inhibitory metal binding site.

Metal exchange is a common strategy to replace the zinc ion of many zinc proteins with other transition metals amenable to spectroscopic investigations. We here demonstrate that in anthrax lethal factor (and likely other zinc proteases), metal exchange is a fast process, and involves the occupation of an inhibitory metal site by the incoming ion prior to the release of zinc.

Recombinant lysine:N(6)-hydroxylase: effect of cysteine-->alanine replacements on structural integrity and catalytic competence.

Recombinant lysine:N(6)-hydroxylase, rIucD, catalyzes the hydroxylation of L-lysine to its N(6)-hydroxy derivative, with NADPH and FAD serving as cofactors in the reaction. The five cysteine residues present in rIucD can be replaced, individually or in combination, with alanine without effecting a major change in the thermal stability, the affinity for L-lysine and FAD, as well as the k(cat) for mono-oxygenase activity of the protein. However, when the susceptibility to modification by either 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 2,6-dichlorophenol indophenol (DPIP) serves as the criterion for monitoring conformational change(s) in rIucD and its muteins, Cys146-->Ala and Cys166-->Ala substitutions are found to induce an enhancement in the reactivity of one of the protein's remaining cysteine residues with concomitant diminution of mono-oxygenase function. In addition, the systematic study of cysteine-->alanine replacement has led to the identification of rIucD's Cys166 as the exposed residue which is detectable during the reaction of the protein with DTNB but not with iodoacetate. Substitution of Cys51 of rIucD with alanine results in an increase in mono-oxygenase activity (approx. 2-fold). Such replacement, unlike those of other cysteine residues, also enables the covalent DPIP conjugate of the protein to accommodate FAD in its catalytic function. A possible role of rIucD's Cys51 in the modulation of its mono-oxygenase function is discussed.

IMP-1 metallo-beta-lactamase: effect of chelators and assessment of metal requirement by electrospray mass spectrometry.

Metallo-beta-lactamases have attracted considerable attention due to their role in microbial resistance to beta-lactam antibiotics. IMP-1, the binuclear Zn-dependent beta-lactamase produced by Pseudomonas aeruginosa and other microorganisms, is of particular interest in view of its increasing prevalence. An examination of the susceptibility of IMP-1 to inactivation by six different divalent metal ion chelators has revealed that all except Zincon cause inhibition by forming a complex with the holoenzyme. Exposure of the enzyme to dipicolinic acid (DPA), the most potent inhibitor, results in the production of the mononuclear Zn form of the protein as determined by electrospray ionization mass spectrometry (ESI-MS) under nondenaturing conditions. This mononuclear Zn species was found to be catalytically competent. Studies with the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) show that the two zinc centers in IMP-1 differ in their accessibility, a feature that could be overcome in the presence of guanidine hydrochloride (GdnHCl, 1.5 M).

Influence of chemical denaturants on the activity, fold and zinc status of anthrax lethal factor.

Anthrax lethal factor (LF) is a zinc-dependent endopeptidase which, through a process facilitated by protective antigen, translocates to the host cell cytosol in a partially unfolded state. In the current report, the influence of urea and guanidine hydrochloride (GdnHCl) on LF׳s catalytic function, fold and metal binding was assessed at neutral pH. Both urea and GdnHCl were found to inhibit LF prior to the onset of unfolding, with the inhibition by the latter denaturant being a consequence of its ionic strength. With the exception of demetallated LF (apoLF) in urea, unfolding, as monitored by tryptophan fluorescence spectroscopy, was found to follow a two-state (native to unfolded) mechanism. Analysis of the metal status of LF with 4-(2-pyridylazoresorcinol) (PAR) following urea or GdnHCl exposure suggests the enzyme to be capable of maintaining its metal ion passed the observed unfolding transition in a chelator-inaccessible form. Although an increase in the concentration of the denaturants eventually allowed the chelator access to the protein׳s zinc ion, such process is not correlated with the release of the metal ion. Indeed, significant dissociation of the zinc ion from LF was not observed even at 6 M urea, and only high concentrations of GdnHCl (>3 M) were capable of inducing the release of the metal ion from the protein. Hence, the current study demonstrates not only the propensity of LF to tightly bind its zinc ion beyond the spectroscopically determined unfolding transition, but also the utility of PAR as a structural probe.

Characterization of the Natural Organic Matter (NOM) in groundwater contaminated with (60)Co and (137)Cs using ultrafiltration, Solid Phase Extraction and fluorescence analysis.

Spot samples of shallow groundwaters have been taken between the years 2004 and 2010 near a site formerly used for the dispersal of radioactive liquid wastes. Three sampling points, one clean (upstream), and two downstream of the contamination source, were processed by ultrafiltration (5000 Da cut-off) and Solid Phase Extraction (SPE) to determine the association of selected artificial radionuclides ((60)Co, (137)Cs) with Natural Organic Matter (NOM). The last two sampling episodes (2008 and 2010) also benefited from fluorescence analysis to determine the major character of the NOM. The fluorescence signals are reported as humic-like, fulvic-like and protein-like, which are used to characterize the different NOM types. The NOM from the clean site comprised mostly fine material, whereas the colloidal content (retained by ultrafiltration) was higher (e.g., 15-40% of the Total Organic Carbon - TOC). Most of the 137Cs was present in the colloidal fraction, whereas (60)Co was found in the filtered fraction. Fluorescence analysis, on the other hand, indicated a contrasting behavior between the clean and contaminated sites, with a dominance of protein-like material, a feature usually associated with human impacts. Finally, SPE removed almost quantitatively the protein-like material (>90%), whereas it removed a much smaller fraction of the (137)Cs (<28%). This finding indicates that the (137)Cs preferential binding occurs with a fraction other than the protein-like NOM, likely the fulvic-like or humic-like portion.