Colorimetric and Fluorimetric Assays to Quantitate Micromolar Concentrations of Transition Metals

McCall, Keith; Fierke, Carol A.

doi:10.1006/abio.2000.4706

Cited by 138 publications

(135 citation statements)

References 42 publications

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“…Protein samples used in all experiments reported were exchanged from the final purification buffer to Hepes 15 mM pH 7.5 using a HiPrep® 26/10 desalting column (Amersham Pharmacia Biotech) or by two dialysis steps against >100 volumes of the same buffer. The Zn(II) content was measured using the colorimetric reagent 4-(2-pyridylazo)-resorcinol (PAR) 19 . Pre steady state kinetic data obtention and analysis.…”

Section: Discussionmentioning

confidence: 99%

Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo-β-lactamase

Rasia¹,

Vila

2003

Arkivoc

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The hydrolysis of the β-lactam compound nitrocefin by the metallo-β-lactamase from B.cereus (BcII) was studied by pre-steady state kinetics measurements, followed by absorbance, fluorescence and diode array detection. In contrast with the results reported for the homologous enzymes CcrA from B.fragilis and L1 from S.maltophilia, no accumulation of an anionic intermediate could be evidenced. The rationale for this observation can be tracked on the lower binding affinity toward a second Zn(II) ion in this enzyme, and the modification of a flexible active site loop, that might contribute to stabilize the anionic intermediate. Analysis of the substrate binding and product formation rates does not support a recently proposed mechanistic scheme that contemplates a nonnegligible accumulation of this intermediate in nitrocefin hydrolysis by BcII.

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Section: Discussionmentioning

confidence: 99%

Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo-β-lactamase

Rasia¹,

Vila

2003

Arkivoc

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“…Samples of the enzyme-free solutions were mixed with 250 M freshly prepared 4-(2-pyridylazo)resorcinol and made up to 1 ml with Chelex (sodium form)-treated buffer. The amount of free Co 2ϩ or Zn 2ϩ present in the reaction was determined in at least duplicate spectrophotometrically at 505 nm for Co 2ϩ and 497 nm for Zn 2ϩ using extinction coefficients described previously (34). The concentration of enzyme-bound Zn 2ϩ or Co 2ϩ was calculated using Equation 1, and dissociation constants were determined by nonlinear regression in GraphPad Prism with Equation 2, where [M 2ϩ ] is concentration of metal and C is the saturated concentration of the metal.…”

Section: Methodsmentioning

confidence: 99%

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

Mazurkewich

Brott

Kimber

et al. 2016

Journal of Biological Chemistry

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The bacterial catabolism of lignin and its breakdown products is of interest for applications in industrial processing of lignobiomass. The gallate degradation pathway of Pseudomonas putida KT2440 requires a 4-carboxy-2-hydroxymuconate (CHM) hydratase (GalB), which has a 12% sequence identity to a previously identified CHM hydratase (LigJ) from Sphingomonas sp. SYK-6. The structure of GalB was determined and found to be a member of the PIG-L N-acetylglucosamine deacetylase family; GalB is structurally distinct from the amidohydrolase fold of LigJ. LigJ has the same stereospecificity as GalB, providing an example of convergent evolution for catalytic conversion of a common metabolite in bacterial aromatic degradation pathways. Purified GalB contains a bound Zn 2؉ cofactor; however the enzyme is capable of using Fe 2؉ and Co 2؉ with similar efficiency. The general base aspartate in the PIG-L deacetylases is an alanine in GalB; replacement of the alanine with aspartate decreased the GalB catalytic efficiency for CHM by 9.5 ؋ 10 4 -fold, and the variant enzyme did not have any detectable hydrolase activity. Kinetic analyses and pH dependence studies of the wild type and variant enzymes suggested roles for Glu-48 and His-164 in the catalytic mechanism. A comparison with the PIG-L deacetylases led to a proposed mechanism for GalB wherein Glu-48 positions and activates the metal-ligated water for the hydration reaction and His-164 acts as a catalytic acid.

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“…For Zn(II) affinity measurements, apo-LpxC C63A (1-200 M) was incubated in a metal ion and pH buffer containing 1 mM nitrilotriacetic acid (NTA), 5 mM Mops, pH 7, 2.5 M dipic-olinic acid, and 0 -0.5 mM Zn tot (0 -3.3 nM Zn free ) at 30°C for 30 min in an anaerobic chamber (39,40). Free and bound metal were separated by ultrafiltration, described above, and metal ions were measured by ICP-MS.…”

Section: Methodsmentioning

confidence: 99%

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

Gattis

Hernick

Fierke

2010

Journal of Biological Chemistry

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UDP-3-O-((R)To analyze the native metal cofactor bound to LpxC, we developed a pulldown method to rapidly purify tagged LpxC under anaerobic conditions. The metal bound to LpxC purified from Escherichia coli grown in minimal medium is mainly Fe(II). However, the ratio of iron/ zinc bound to LpxC varies with the metal content of the medium. Furthermore, the iron/zinc ratio bound to native LpxC, determined by activity assays, has a similar dependence on the growth conditions. LpxC has significantly higher affinity for Zn(II) compared with Fe(II) with K D values of 60 ؎ 20 pM and 110 ؎ 40 nM, respectively. However, in vivo concentrations of readily exchangeable iron are significantly higher than zinc, suggesting that Fe(II) is the thermodynamically favored metal cofactor for LpxC under cellular conditions. These data indicate that LpxC expressed in E. coli grown in standard medium predominantly exists as the Fe(II)-enzyme. However, the metal cofactor in LpxC can switch between iron and zinc in response to perturbations in available metal ions. This alteration may be important for regulating the LpxC activity upon changes in environmental conditions and may be a general mechanism of regulating the activity of metalloenzymes.Gram-negative bacteria are important targets in the continuing fight against antibiotic-resistant infections. The development of new antibiotics to treat infections caused by resistant organisms is critically needed and will require novel targets. One potential source of targets in Gram-negative bacteria is the lipid A biosynthetic pathway (Fig. 1A) (1, 2), an essential building block of lipopolysaccharides (LPS) that make up the outer leaflet surrounding the cell wall in Gram-negative bacteria (2-4). In addition to being essential for cell viability, LPS are also referred to as endotoxins (2, 4, 5) and are responsible for immunogenic stimulation in septic shock that can lead to a number of deleterious effects, including severe hypotension, multiple organ dysfunction, and death (5). Consequently, inhibitors of lipid A biosynthesis have the potential to serve as both antibiotics and antiendotoxins (6). The UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) 3 catalyzes the committed and second overall step in lipid A biosynthesis, the hydrolysis of UDP-3-O-myristoyl-N-acetylglucosamine to UDP-3-O-myristoyl-glucosamine and acetate ( Fig. 1) (7-10). Consequently, inhibitors of LpxC are an active area of drug development (11)(12)(13)(14).LpxC was previously identified as a mononuclear Zn(II) metalloenzyme (Fig. 1B) (15) that also contains a weaker, inhibitory metal ion-binding site. This catalytic metal ion binds and polarizes an inner sphere water molecule that is activated by general base catalysis to serve as the nucleophile for hydrolysis of the acetyl substrate (Fig. 1C) (16, 17). However, recent evidence has shown that LpxC is more active with Fe(II) as its metal ion cofactor compared with Zn(II) (18). This increase in activity may correlate with a change in the ground stat...

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Colorimetric and Fluorimetric Assays to Quantitate Micromolar Concentrations of Transition Metals

Cited by 138 publications

References 42 publications

Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo-β-lactamase

Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo-β-lactamase

Structural and Kinetic Characterization of the 4-Carboxy-2-hydroxymuconate Hydratase from the Gallate and Protocatechuate 4,5-Cleavage Pathways of Pseudomonas putida KT2440

Active Site Metal Ion in UDP-3-O-((R)-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase (LpxC) Switches between Fe(II) and Zn(II) Depending on Cellular Conditions*

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