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Articles 61 - 81 of 81
Full-Text Articles in Physical Sciences and Mathematics
Analyzing The Catalytic Mechanism Of The Fe-Type Nitrile Hydratase From Comamonas Testosteroni Ni1, Saroja Rao, Richard Holz
Analyzing The Catalytic Mechanism Of The Fe-Type Nitrile Hydratase From Comamonas Testosteroni Ni1, Saroja Rao, Richard Holz
Richard C. Holz
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 …
Function Of The Signal Peptide And N- And C-Terminal Propeptides In The Leucine Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Ventris D'Souza, Guanjing Chen, Heidi Campbell, Alice Mitchell, Richard Holz
Function Of The Signal Peptide And N- And C-Terminal Propeptides In The Leucine Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Ventris D'Souza, Guanjing Chen, Heidi Campbell, Alice Mitchell, Richard Holz
Richard C. Holz
The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type …
Divalent Metal Binding Properties Of The Methionyl Aminopeptidase From Escherichia Coli, Ventris D'Souza, Brian Bennett, Alicja Copik, Richard Holz
Divalent Metal Binding Properties Of The Methionyl Aminopeptidase From Escherichia Coli, Ventris D'Souza, Brian Bennett, Alicja Copik, Richard Holz
Richard C. Holz
The metal-binding properties of the methionyl aminopeptidase from Escherichia coli (MetAP) were investigated. Measurements of catalytic activity as a function of added Co(II) and Fe(II) revealed that maximal enzymatic activity is observed after the addition of only 1 equiv of divalent metal ion. Based on these studies, metal binding constants for the first metal binding event were found to be 0.3 ± 0.2 μM and 0.2 ± 0.2 μM for Co(II)- and Fe(II)-substituted MetAP, respectively. Binding of excess metal ions (>50 equiv) resulted in the loss of ∼50% of the catalytic activity. Electronic absorption spectral titration of a 1 …
The Proteomics Of N-Terminal Methionine Cleavage, Frédéric Frottin, Aude Martinez, Philippe Peynot, Sanghamitra Mitra, Richard Holz, Carmela Giglione, Thierry Meinnel
The Proteomics Of N-Terminal Methionine Cleavage, Frédéric Frottin, Aude Martinez, Philippe Peynot, Sanghamitra Mitra, Richard Holz, Carmela Giglione, Thierry Meinnel
Richard C. Holz
Methionine aminopeptidase (MAP) is a ubiquitous, essential enzyme involved in protein N-terminal methionine excision. According to the generally accepted cleavage rules for MAP, this enzyme cleaves all proteins with small side chains on the residue in the second position (P1′), but many exceptions are known. The substrate specificity of Escherichia coli MAP1 was studied in vitro with a large (>120) coherent array of peptides mimicking the natural substrates and kinetically analyzed in detail. Peptides with Val or Thr at P1′ were much less efficiently cleaved than those with Ala, Cys, Gly, Pro, or Ser in this position. Certain residues …
Unraveling The Catalytic Mechanism Of Nitrile Hydratases, Sanghamitra Mitra, Richard Holz
Unraveling The Catalytic Mechanism Of Nitrile Hydratases, Sanghamitra Mitra, Richard Holz
Richard C. Holz
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 ± …
Proton Nmr Investigation Of The [4fe-4s]1+ Cluster Environment Of Nitrogenase Iron Protein From Azotobacter Vinelandii: Defining Nucleotide-Induced Conformational Changes, William Lanzilotta, Richard Holz, Lance Seefeldt
Proton Nmr Investigation Of The [4fe-4s]1+ Cluster Environment Of Nitrogenase Iron Protein From Azotobacter Vinelandii: Defining Nucleotide-Induced Conformational Changes, William Lanzilotta, Richard Holz, Lance Seefeldt
Richard C. Holz
This work presents the complete assignment of the isotropically shifted 1H NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to β-CH2 and α-CH protons of the [4Fe- 4S]1+ cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T1 measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand β-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand α-CH protons. Temperature …
Hydrolysis Of Thionopeptides By The Aminopeptidase From Aeromonas Proteolytica: Insight Into Substrate Binding, David Bienvenue, Danuta Gilner, Richard Holz
Hydrolysis Of Thionopeptides By The Aminopeptidase From Aeromonas Proteolytica: Insight Into Substrate Binding, David Bienvenue, Danuta Gilner, Richard Holz
Richard C. Holz
A series of l-leucine aniline analogues were synthesized that contained either a carbonyl or thiocarbonyl as a part of the amide bond. Additionally, the para-position on the phenyl ring of several substrates was altered with various electron-withdrawing or donating groups. The kinetic constants Km and kcat were determined for the hydrolysis of each of these compounds in the presence of the aminopeptidase from Aeromonas proteolytica (AAP) containing either Zn(II) or Cd(II). The dizinc(II) form of AAP ([ZnZn(AAP)]) was able to cleave both carbonyl and thiocarbonyl containing peptide substrates with similar efficiency. However, the dicadmium(II) form of AAP ([CdCd(AAP)]) was unable …
Electrochemical Attachment Of Motile Bacterial Cells To Gold, Sergey Rozhok, Richard Holz
Electrochemical Attachment Of Motile Bacterial Cells To Gold, Sergey Rozhok, Richard Holz
Richard C. Holz
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 …
Synthesis, Molecular Structure, And Reactivity Of Dinuclear Copper(Ii) Complexes With Carboxylate-Rich Coordination Environments, Richard Holz, John Bradshaw, Brian Bennett
Synthesis, Molecular Structure, And Reactivity Of Dinuclear Copper(Ii) Complexes With Carboxylate-Rich Coordination Environments, Richard Holz, John Bradshaw, Brian Bennett
Richard C. Holz
The dinucleating ligand N,N‘-(2-hydroxy-5-methyl-1,3-xylylene)bis(N-(carboxymethyl)glycine) (CH3HXTA) has been used to synthesize the dinuclear Cu(II) bis(pyridine) complex Na[Cu2(CH3HXTA)(Py)2]·1.5(1,4-dioxane) (Na(1)): triclinic space group P1̄ (a = 12.550(3) Å, b = 13.413(3) Å, c = 13.540(4) Å, α = 117.12(2)°, β = 104.70 (2)°, and γ = 92.13(2)°). The structure shows two distinct distorted square pyramidal Cu(II) centers with each Cu(II) ion bound by two carboxylate oxygen atoms, one amine nitrogen atom, a phenolate oxygen atom, and one pyridine nitrogen atom. The Cu--Cu separation is 3.531 Å, …
Structurally Distinct Active Sites In The Copper(Ii)-Substituted Aminopeptidases From Aeromonas Proteolytica And Escherichia Coli, Brian Bennett, William E. Antholine, Ventris M. D'Souza, Guanjing Chen, Leila Ustinyuk, Richard C. Holz
Structurally Distinct Active Sites In The Copper(Ii)-Substituted Aminopeptidases From Aeromonas Proteolytica And Escherichia Coli, Brian Bennett, William E. Antholine, Ventris M. D'Souza, Guanjing Chen, Leila Ustinyuk, Richard C. Holz
Richard C. Holz
The aminopeptidase from Aeromonas proteolytica (AAP) was titrated with copper, which bound sequentially at two distinct sites. Both the mono- and disubstituted forms of AAP exhibited catalytic hyperactivity relative to the native dizinc enzyme. Monosubstituted AAP exhibited an axial Cu(II) EPR spectrum with slight pH dependence: at pH 6.0 g|| = 2.249, g⊥ = 2.055, and A||(63/65Cu) = 1.77 × 10-2 cm-1, whereas at pH 9.65 g|| = 2.245, g⊥ = 2.056, and A||(63/65Cu) = 1.77 × 10-2 cm-1. These data indicate oxygen and nitrogen ligation of Cu. AAP further substituted with copper exhibited a complex signal with features around …
Proton Nmr Spectroscopy As A Probe Of Dinuclear Copper(Ii) Centers, Richard Holz, Julie Brink
Proton Nmr Spectroscopy As A Probe Of Dinuclear Copper(Ii) Centers, Richard Holz, Julie Brink
Richard C. Holz
No abstract provided.
Dinuclear Copper(Ii) Complexes With Carboxylate-Rich Coordination Environments. Models For Substituted Copper(Ii) Aminopeptidases, Richard Holz, Julie Brink, Feben Gobena, Charles O'Connor
Dinuclear Copper(Ii) Complexes With Carboxylate-Rich Coordination Environments. Models For Substituted Copper(Ii) Aminopeptidases, Richard Holz, Julie Brink, Feben Gobena, Charles O'Connor
Richard C. Holz
The dinucleating ligand N,N'-(2-hydroxy-5-methyl1-, 3-xylylene)bis(N-carboxymethylglycine) (CH3HXTA) has been used to synthesize the dinuclear Cu(II) diaqua complex [Cu2(CH3HXTA) (H2O)2]H•4H2O (1): monoclinic space group P21In (a = 15.092(3) Å, b = 10.842(3) Å, c = 15.219(4) Å, and β = 104.70(2)°). The structure shows two distinct square pyramidal Cu(II) centers with each Cu(II) ion bound to two carboxylate oxygen atoms, one amine nitrogen atom, the phenolate oxygen atom, and one water oxygen atom. The Cu- -Cu separation is 3.726 Å and Cu1-O1-Cu2 angle …
Structures And Properties Of Dibridged (Μ-Oxo)Diiron(Iii) Complexes. Effects Of The Fe-O-Fe Angle, Richard Norman, Richard Holz, Stephane Menage, Charles O'Connor, Jian Zhang, Lawrence Que
Structures And Properties Of Dibridged (Μ-Oxo)Diiron(Iii) Complexes. Effects Of The Fe-O-Fe Angle, Richard Norman, Richard Holz, Stephane Menage, Charles O'Connor, Jian Zhang, Lawrence Que
Richard C. Holz
A series of (μ-oxo)diiron(lll) complexes of tris(2-pyridylmethy1)amine (TPA), [Fe2(TPA)2O(L)] (CIO4)n, were synthesized and characterized where L represents the bridging ligands carbonate, hydrogen maleate, diphenyl phosphate, diphenylphosphinate, maleate, and phthalate. Together with the linear dichloride complex, this series of compounds provides a unique opportunity to systematically study the effects of the Fe-0-Fe angle (125-180°) on the electronic spectral and magnetic properties of the (μ-oxo)diiron(lll) core. [Fe2(TPA)2O(CO3)](CIO4)2.2CH3OH (1) crystallizes in the monoclinic space group P21/c with a = 11.282 (7) Å, b = 18.253 (9) Å, c = 20.390 (7) Å, and β= 95.02 (4)°. The structure was determined at -50°C from …
Attachment Of Motile Bacterial Cells To Prealigned Holed Microarrays, Sergey Rozhok, Zhifang Fan, Dorjderem Nyamjav, Chang Liu, Chad Mirkin, Richard Holz
Attachment Of Motile Bacterial Cells To Prealigned Holed Microarrays, Sergey Rozhok, Zhifang Fan, Dorjderem Nyamjav, Chang Liu, Chad Mirkin, Richard Holz
Richard C. Holz
Construction of biomotors is an exciting area of scientific research that holds great promise for the development of new technologies with broad potential applications in areas such as the energy industry and medicine. Herein, we demonstrate the fabrication of prealigned microarrays of motile Escherichia coli bacterial cells on SiOx substrates. To prepare these arrays, holed surfaces with a gold layer on the bottom of the holes were utilized. The attachment of bacteria to the holes was achieved via nonspecific interactions using poly-l-lysine hydrobromide (PLL). Our data suggest that a single motile bacterial cell can be selectively attached to an individual …
Two-Dimensional 1H Nmr Studies On Octahedral Nickel(Ii) Complexes, Richard Holz, Evgenij Evdokimov, Feben Gobena
Two-Dimensional 1H Nmr Studies On Octahedral Nickel(Ii) Complexes, Richard Holz, Evgenij Evdokimov, Feben Gobena
Richard C. Holz
The dinucleating ligand ethylene glycol−bis(β-aminoethyl ether) N,N,N‘,N‘-tetrakis[(2-(1-ethylbenzimidazoyl)] (EGTB-Et; 1) was used to synthesize the dinuclear Ni(II) tetraacetonitrile complex cation [Ni2(EGTB-Et)(CH3CN)4]2+ (2): triclinic space group P1̄ (a = 12.273(5) Å, b = 12.358(7) Å, c = 12.561(6) Å, α = 90.43(4)°, β = 110.26(3)°, γ = 99.21 (4)°, and Z = 1). The structure shows two identical octahedral Ni(II) centers each bound to two benzimidazole ring nitrogen atoms, one amine nitrogen atom, an ether oxygen atom, and two acetonitrile nitrogen atoms. The Ni(II) ions are tethered together by a diethyl ether linkage with a crystallographic center of inversion between the methylene …
Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli Contains A Dinuclear Metalloactive Site, Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli Contains A Dinuclear Metalloactive Site, Wade C. Mcgregor, Sabina I. Swierczek, Brian Bennett, Richard C. Holz
Richard C. Holz
The catalytic and structural properties of the argE-encoded N-acetyl-l-ornithine deacetylase (ArgE) from Escherichia coli were investigated. On the basis of kinetic and ITC (isothermal titration calorimetry) data, Zn(II) binds to ArgE with Kd values that differ by ∼20 times. Moreover, ArgE exhibits ∼90% of its full catalytic activity upon addition of one metal ion. Therefore, ArgE behaves similarly to the aminopeptidase from Aeromonas proteolytica (AAP) in that one metal ion is the catalytic metal ion while the second likely plays a structural role. The N-acetyl-l-ornithine (NAO) deacetylase activity of ArgE showed a linear temperature dependence from 20 to 45 °C, …
Proton Nuclear Magnetic Resonance Investigation Of The [2fe-2s]1--Containing “Rieske-Type” Protein From Xanthobacter Strain Py2, Richard Holz, Frederick Small, Scott Ensign
Proton Nuclear Magnetic Resonance Investigation Of The [2fe-2s]1--Containing “Rieske-Type” Protein From Xanthobacter Strain Py2, Richard Holz, Frederick Small, Scott Ensign
Richard C. Holz
Proton NMR spectra of the Rieske-type ferredoxin from Xanthobacter strain Py2 were recorded in both H2O and D2O buffered solutions at pH 7.2. Several well-resolved hyperfine-shifted 1H NMR signals were observed in the 90 to −20 ppm chemical shift range. Comparison of spectra recorded in H2O and D2O buffered solutions indicated that the signals at −11.4 (L) and −15.5 (M) ppm were solvent-exchangeable and thus were assigned to the two histidine Nε2H protons. The remaining observed signals were assigned based upon chemical shift, T1 values, and one-dimensional nuclear Overhauser effect (nOe) saturation transfer experiments to either CβH or CαH protons …
Spectroscopic Characterization Of Some Rare Earth Complexes Of Triethylenetetraaminehexaacetic Acid, Richard Holz, William Horrocks
Spectroscopic Characterization Of Some Rare Earth Complexes Of Triethylenetetraaminehexaacetic Acid, Richard Holz, William Horrocks
Richard C. Holz
The Eu3+ complexes formed with triethylenetetraaminehexaacetic acid (TTHA) were characterized in solution by laser-induced Eu3+ luminescence spectroscopy. Both 2:1 and 1:1 metal:ligand complexes were detected at pH 6.0 depending on the metal- ligand stoichiometry. These two species exhibit distinct excited-state lifetimes of 260 and 1210 μs, respectively. The 2:1 complex coordinates three or four water molecules at each Eu3+ ion while the 1:1 complex has no coordinated water molecules. The 7F0→5D0 excitation spectra revealed multiple isomeric forms for both the 2:1 and 1:1 complexes. Spectra and excited-state lifetimes were taken as a function of pH for a 1:1 metal:ligand solution. …
Yttrium-89 Nmr Spectroscopy, A New Probe For Calcium-Binding Proteins, Richard Holz, William Horrocks
Yttrium-89 Nmr Spectroscopy, A New Probe For Calcium-Binding Proteins, Richard Holz, William Horrocks
Richard C. Holz
No abstract provided.
Molecular Discrimination Of Type-I Over Type-Ii Methionyl Aminopeptidases, Krzysztof Swierczek, Alicja Copik, Sabina Swierczek, Richard Holz
Molecular Discrimination Of Type-I Over Type-Ii Methionyl Aminopeptidases, Krzysztof Swierczek, Alicja Copik, Sabina Swierczek, Richard Holz
Richard C. Holz
Two residues that are conserved in type-I methionyl aminopeptidases (MetAPs) but are absent in all type-II MetAPs are the cysteine residues (Escherichia coli MetAP-I: C59 and C70) that reside at the back of the substrate recognition pocket. These Cys residues are 4.4 Å apart and do not form a disulfide bond. Since bacteria and fungi contain only type-I MetAPs while all human cells contain both type-I and type-II MetAPs, type-I MetAPs represent a novel antibiotic/antifungal target if type-I MetAPs can be specifically targeted over type-II. Based on reaction of the thiol-specific binding reagent 5,5‘-dithio-bis(2-nitrobenzoic acid) (DTNB) with the type-I MetAP …
Zinc Aminopeptidases, Aminopeptidase From Vibrio Proteolyticus (Aeromonas Proteolytica) As Prototypical Enzyme, Richard Holz, Anna Starus, Danuta Gillner
Zinc Aminopeptidases, Aminopeptidase From Vibrio Proteolyticus (Aeromonas Proteolytica) As Prototypical Enzyme, Richard Holz, Anna Starus, Danuta Gillner
Richard C. Holz
No abstract provided.