421 research outputs found
Analyzing the Catalytic Mechanism of the Fe-Type Nitrile Hydratase from \u3cem\u3eComamonas testosteroni\u3c/em\u3e Ni1
In order to gain insight into the catalytic mechanism of Fe-type nitrile hydratases (NHase), the pH and temperature dependence of the kinetic parameters kcat, Km, and kcat/Km along with the solvent isotope effect were examined for the Fe-type NHase from Comamonas testosteroni Ni1 (CtNHase). CtNHase was found to exhibit a bell-shaped curve for plots of relative activity vs pH over pH values 4−10 for the hydration of acrylonitrile and was found to display maximal activity at pH ∼7.2. Fits of these data provided a pKES1 value of 6.1 ± 0.1, a pKES2 value of 9.1 ± 0.2 (k′cat = 10.1 ± 0.3 s−1), a pKE1 value of 6.2 ± 0.1, and a pKE2 value of 9.2 ± 0.1 (k′cat/K′m of 2.0 ± 0.2 s−1 mM−1). Proton inventory studies indicate that two protons are transferred in the rate-limiting step of the reaction at pH 7.2. Since CtNHase is stable to 25 °C, an Arrhenius plot was constructed by plotting ln(kcat) vs 1/T, providing an Ea of 33.3 ± 1.5 kJ/mol. ΔH° of ionization values were also determined, thus helping to identify the ionizing groups exhibiting the pKES1 and pKES2 values. Based on ΔH°ion data, pKES1 is assigned to βTyr68 while pKES2 is assigned to βArg52, βArg157, or αSer116 (NHases are α2β2 heterotetramers). Given the strong similarities in the kinetic data obtained for both Co- and Fe-type NHase enzymes, both types of NHase enzymes likely hydrate nitriles in a similar fashion
Electrochemical Attachment of Motile Bacterial Cells to Gold
Selective attachment of Escherichia coli K-12 bacterial cells to charged gold surfaces was demonstrated. Electrostatic binding of E. coli K-12 bacterial cells to positively charged surfaces was observed starting at +750 mV. The binding of E. coli K-12 cells to positively charged gold surfaces is proposed to occur due to long-range electrostatic interactions between the negatively charged O-chain of lipopolysaccharide (LPS) molecules protruding the bacterial cell body and the electrode surface. Removing LPS alters the cellular surface charge and results in cellular attachment to negatively charged surfaces. Thus, applying an electrical potential allows for the direct, real time detection of live, dead or damaged bacterial cells. The attachment of E. coli K-12 bacterial cells to surfaces with an applied potential substantiates the hypothesis that an electrostatic interaction is responsible for the binding of bacterial cells to positively charged molecular assemblies on surfaces used for building bacterial microarrays
Spectroscopically Distinct Cobalt(II) Sites in Heterodimetallic Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e:  Characterization of Substrate Binding
The Co(II)Zn(II)- and Zn(II)Co(II)-substituted derivatives of the aminopeptidase from Aeromonas proteolytica (AAP) were probed by EPR spectroscopy. EPR spectra of the high-spin S = 3/2 Co(II) ions in [CoZn(AAP)] and [ZnCo(AAP)] indicated that each metal binding site provides a spectroscopically distinct signature. For [CoZn(AAP)], subtraction of EPR spectra recorded at pH 7.5 and 10 revealed that two species were present and that the relative contributions to each of the experimental spectra were pH-dependent. The first EPR species, predominant at lower pH values, was simulated as a relatively featureless axial signal with geff values of 2.20, 3.92, and 5.23 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.29 and an E/D of 0.1. The second species, predominant at high pH, was simulated with geff values of 1.80, 2.75, and 6.88 and exhibited a characteristic eight-line 59Co hyperfine pattern with an Az(59Co) of 7.0 mT. These parameters correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.54; however, the signal exhibited marked rhombicity (E/D = 0.32) indicative of an asymmetric tetrahedral or five-coordinate Co(II) ion. Summation of these two species provided an excellent simulation of the observed [CoZn(AAP)] EPR spectrum. The EPR spectrum of [ZnCo(AAP)] also contained two species, at least one of which also exhibited 59Co hyperfine features. However, this signal exhibited little pH dependence, and individual species could not be isolated. The addition of the competitive inhibitor 1-butaneboronic acid (BuBA) to [CoZn(AAP)] resulted in a distinct change in the EPR spectrum; however, addition of BuBA to [ZnCo(AAP)] left the EPR spectrum completely unperturbed. These data indicate that BuBA binds only to the first metal binding site in AAP and does not interact with the second site. On the basis of the X-ray crystallographic data for the transition state analog-inhibited complexes of AAP and the aminopeptidase from bovine lens, BuBA was reclassified as a substrate analog inhibitor rather than a transition state analog inhibitor as previously suggested [Baker, J. O., & Prescott, J. M. (1983) Biochemistry 22, 5322−5331]. From difference spectroscopy and from the simulation of the [CoZn(AAP)] EPR spectrum, a third signal appearing upon BuBA binding was isolated. This signal was simulated with geff values of 2.08, 3.15, and 6.15 which correspond to an Ms = |±1/2〉 ground state transition with a greal of 2.41 and an E/D of 0.22. This simulation also invoked an eight-line unresolved 59Co hyperfine pattern with an Az(59Co) value of 4.0 mT. Summation of the these three species provided an excellent simulation of the observed [CoZn(AAP)] + BuBA EPR spectrum at both pH values. This work establishes that substrate binds only to the first metal binding site in AAP and thus substantiates the first step in catalysis in the recently proposed mechanism of action for AAP [Bennett, B., & Holz, R. C. (1997) J. Am. Chem. Soc. 119, 1923−1933; Chen, G., et al. (1997) Biochemistry 36, 4278−4286]
Unraveling the Catalytic Mechanism of Nitrile Hydratases
To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants kcat and Km for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2–11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pKES1 and pKES2 values of 5.9 ± 0.1 and 9.2 ± 0.1 (kcat′ = 130 ± 1 s-1), respectively, and pKE1 and pKE2 values of 5.8 ± 0.1 and 9.1 ± 0.1 (kcat′/Km′ = (6.5 ± 0.1) × 103 s-1 mm-1), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 °C, an Arrhenius plot was constructed by plotting ln(kcat) versus 1/T, providing Ea = 23.0 ± 1.2 kJ/mol. The thermal stability of PtNHase also allowed ΔH0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pKES1 and pKES2 values. Based on ΔH0ion data, pKES1 is assigned to βTyr68, whereas pKES2 is assigned to βArg52, βArg157, or αSer112 (NHases are α2β2-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed
EPR Studies on the Mono- and Dicobalt(II)-Substituted Forms of the Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e. Insight into the Catalytic Mechanism of Dinuclear Hydrolases
The structure and function of the prototypical dinuclear hydrolase, namely, the aminopeptidase from Aeromonas proteolytica (AAP), was probed by EPR spectroscopy of the mono- and dicobalt(II)-substituted derivatives. A new systematic protocol for the interpretation of Co(II) EPR spectra is described and the S = 3/2 spin states of the Co(II)-substituted forms of the enzyme have been characterized. This protocol allows the simulation of line shape using theoretically allowed geff values corresponding to an isotropic greal value. In addition, the gross distortion of EPR spectra of high-spin S = 3/2 Co(II) ions has been investigated, and the effects of saturation on the line shapes and on simulation-derived spectral parameters are discussed. For [Co-(AAP)], a distinctive EPR signal was observed in which the hyperfine pattern due to 59Co was not centered on the low-field absorption feature, and the signal could not be simulated as a single species. Subtraction of EPR spectra recorded at different temperatures revealed that two species were, in fact, present in samples of [Co-(AAP)]. The first species was a relatively featureless axial signal with geff values of 5.75, 4.50, and 2.50. These values correspond to an Ms = |±1/2〉 ground-state transition with greal = 2.57 and E/D = 0.08. The second species had geff values of 6.83, 2.95, and 1.96 and exhibited a characteristic eight-line 59Co hyperfine pattern with Az = 7.2 mT. The observed 59Co hyperfine lines were simulated in both line width as well as signal intensity for the first time. These parameters correspond to the Ms = |±1/2〉 ground-state transition with greal = 2.57; however, the signal exhibited marked rhombicity (E/D = 0.28), consistent with a highly distorted tetrahedral Co(II) species. The possibility that the spectrum could be due to contributions from the Ms = |±1/2〉 and Ms = |±3/2〉 doublets of a single spin system was investigated, but subtraction of spectra recorded at various temperatures clearly indicated that the features at g = 2.95 and g = 1.96 were correlated with the feature at g = 6.83. In addition, at temperatures above 15 K, the signal intensity rapidly decreases and the signal is lost. The EPR spectrum of [CoCo(AAP)] reveals a relatively featureless signal that was simulated as a single species with geff(1,2,3) values of 5.10, 3.85, and 2.19; Ms = |±1/2〉; greal = 2.25; E/D = 0.095. The intensity of the observed signal for [CoCo(AAP)] corresponded to 0.13 spin/mol of Co(II). These data strongly suggest that the two Co(II) ions in the active site of AAP experience significant spin−spin interaction and are either antiferromagnetically or ferromagnetically coupled. Perpendicular mode EPR titration of apo-AAP with Co(II) revealed a low-field signal extending out of zero-field in samples with more than 1 equiv of Co(II) added. This type of EPR absorption is indicative of an integral spin system. Coincident with the appearance of the low-field perpendicular mode signal was the appearance of a parallel mode EPR signal with g ∼ 12. These data represent the first definitive evidence for ferromagnetic coupling between two high-spin S = 3/2 Co(II) ions in a dinuclear center. The effect of pH, added peroxide, and the coordination of the competitive inhibitor 1-butaneboronic acid (BuBA) on the signal both confirm the origin of the signal and provide important mechanistic information for this novel dicobalt(II) active site cluster. Based on the present study and the available literature data, a detailed mechanism of action is proposed for AAP
The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from \u3cem\u3eAeromonas proteolytica\u3c/em\u3e
Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (Km) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants kcat and Km revealed a change in the pKa of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pKa values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base
Structure of 2,9-dimethyl-1,10-phenanthroline hemihydrate
C14H12N2•½H2O, Mr = 217•27, tetragonal I41/a, a = 14•258 (3), c = 22•286 (4) Å, V = 4531 (3) Å3, Z = 16, Dx = 1•274 (1) g cm-3, Mo Kα radiation λ = 0•71073 Å, µ = 0•74 cm-1, F(000) = 1840, T = 297 K, R = 0•041 for 1196 unique observed reflections with I \u3e 2σ(I). Pairs of dimethylphenanthroline molecules related by a twofold axis are bridged by water molecules lying on the twofold axis and H bonded to one of the N atoms in each molecule. The H bonds are long and far from linear: O—H 1•06 (4), H•••N 154 (3)°. This is presumably a consequence of the approximately parallel arrangement of the two phenanthroline molecules in the (phen)2.H2O complex, which are tilted 4•7 (1)° with respect to each other; the atoms in one molecule are 3•50 to 3•81 Å from the plane of the other molecule. On the other side of the phenanthroline is another phenanthroline related by a center of symmetry with the atoms of one molecule 3•41 to 3•45 Å from the plane of the other molecule. The phenanthroline molecule has close to 2mm symmetry, but the individual C6 rings are tilted about 1° with respect to each other
Immobilization of Motile Bacterial Cells via Dip-pen Nanolithography
A strategy to bind bacterial cells to surfaces in a directed fashion via dip-pen nanolithography (DPN) is presented. Cellular attachment to pre-designed DPN generated microarrays was found to be dependent on the shape and size of the surface feature. While this observation is likely due in part to a dense, well formed mercaptohexadecanoic acid (MHA) monolayer generated via DPN, it may also simply be due to the physical shape of the surface structure. Motile Pseudomonas aeruginosa bacterial cells were observed to bind to DPN generated mercaptohexadecanoic acid/poly-L-lysine (MHA/PLL) line patterns, \u27blocks\u27 made up of eight lines with 100 nm spacings, with ~ 80% occupancy. Cellular binding to these \u27block\u27 surface structures occurs via an electrostatic interaction between negatively charged groups on the bacterial cell surface and positively charged poly-L-lysine (PLL) assemblies. These data indicate that these DPN generated \u27block\u27 surface structures provide a promising footprint for the attachment of motile bacterial cells that may find utility in cell based biosensors or single cell studies
- …