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Articles 511 - 540 of 2524

Full-Text Articles in Physical Sciences and Mathematics

Structural Evidence That The Methionyl Aminopeptidase From Escherichia Coli Is A Mononuclear Metalloprotease, Nathaniel Cosper, Ventris D'Souza, Robert Scott, Richard Holz Mar 2015

Structural Evidence That The Methionyl Aminopeptidase From Escherichia Coli Is A Mononuclear Metalloprotease, Nathaniel Cosper, Ventris D'Souza, Robert Scott, Richard Holz

Richard C. Holz

The Co and Fe K-edge extended X-ray absorption fine structure (EXAFS) spectra of the methionyl aminopeptidase from Escherichia coli (EcMetAP) have been recorded in the presence of 1 and 2 equiv of either Co(II) or Fe(II) (i.e., [Co(II)_(EcMetAP)], [Co(II)Co(II)(EcMetAP)], [Fe(II)_(EcMetAP)], and [Fe(II)Fe(II)(EcMetAP)]). The Fourier transformed data of both [Co(II)_(EcMetAP)] and [Co(II)Co(II)(EcMetAP)] are dominated by a peak at ca. 2.05 Å, which can be fit assuming 5 light atom (N,O) scatterers at 2.04 Å. Attempts to include a Co−Co interaction (in the 2.4−4.0 Å range) in the curve-fitting parameters were unsuccessful. Inclusion of multiple-scattering contributions from the outer-shell atoms of a …

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Characterization Of The Active Site And Insight Into The Binding Mode Of The Anti-Angiogenesis Agent Fumagillin To The Manganese(Ii)-Loaded Methionyl Aminopeptidase From Escherichia Coli, Ventris D'Souza, Robert Brown, Brian Bennett, Richard Holz Mar 2015

Characterization Of The Active Site And Insight Into The Binding Mode Of The Anti-Angiogenesis Agent Fumagillin To The Manganese(Ii)-Loaded Methionyl Aminopeptidase From Escherichia Coli, Ventris D'Souza, Robert Brown, Brian Bennett, Richard Holz

Richard C. Holz

EPR spectra were recorded for methionine aminopeptidase from Escherichia coli (EcMetAP-I) samples (~2.5 mM) to which one and two equivalents of Mn(II) were added (the latter is referred to as [MnMn(EcMetAP-I)]). The spectra for each sample were indistinguishable except that the spectrum of [MnMn(EcMetAP-I)] was twice as intense. The EPR spectrum of [MnMn(EcMetAP-I)] exhibited the characteristic six-line g≈2 EPR signal of mononuclear Mn(II) with A av(55Mn)=9.3 mT (93 G) and exhibited Curie-law temperature dependence. This signal is typical of Mn(II) in a ligand sphere comprising oxygen and/or nitrogen …

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Exafs Evidence For A "Cysteine Switch" In The Activation Of Prostromelysin, Richard Holz, Scott Salowe, Catherine Smith, Gregory Cuca, Lawrence Que Mar 2015

Exafs Evidence For A "Cysteine Switch" In The Activation Of Prostromelysin, Richard Holz, Scott Salowe, Catherine Smith, Gregory Cuca, Lawrence Que

Richard C. Holz

Zn K-edge EXAFS data of the matrix metalloproteinase (MMP) stromelysin-1 were obtained in both its latent proenzyme and mature active forms. The Fourier-filtered (back-transform 0.7-2.3 Å) xk3 spectrum of mature stromelysin was satisfactorily simulated with 4 N / O scatterers per Zn at 2.01 Å, while similar fits for prostromelysin were judged unacceptable because of unreasonable Debye-Waller factors or significantly larger residuals of the fits. For prostromelysin, excellent fits were obtained with the introduction of a sulfur scatterer at 2.25 Å. These data provide the first direct evidence for the coordination of zinc by the sole cysteine in the N-terminal …

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Methods For Fabricating Microarrays Of Motile Bacteria, Sergey Rozhok, Clifton Shen, Pey-Lih Littler, Zhifang Fan, Chang Liu, Chad Mirkin, Richard Holz Mar 2015

Methods For Fabricating Microarrays Of Motile Bacteria, Sergey Rozhok, Clifton Shen, Pey-Lih Littler, Zhifang Fan, Chang Liu, Chad Mirkin, Richard Holz

Richard C. Holz

Motile bacterial cell microarrays were fabricated by attaching Escherichia coli K-12 cells onto predesigned 16-mercaptohexadecanoic acid patterned microarrays, which were covalently functionalized with E. coli antibodies or poly-L-lysine. By utilizing 11-mercaptoundecyl-penta(ethylene glycol) or 11-mercapto-1-undecanol as passivating molecules, nonspecific binding of E. coli was significantly reduced. Microcontact printing and dip-pen nanolithography were used to prepare microarrays for bacterial adhesion, which was studied by optical fluorescence and atomic force microscopy. These data indicate that single motile E. coli can be attached to predesigned line or dot features and binding can occur via the cell body or the flagella of bacteria. Adherent bacteria …

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Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By L-Leucinethiol: Kinetic And Spectroscopic Characterization Of A Slow, Tight-Binding Inhibitor–Enzyme Complex, David Bienvenue, Brian Bennett, Richard Holz Mar 2015

Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By L-Leucinethiol: Kinetic And Spectroscopic Characterization Of A Slow, Tight-Binding Inhibitor–Enzyme Complex, David Bienvenue, Brian Bennett, Richard Holz

Richard C. Holz

The peptide inhibitor l-leucinethiol (LeuSH) was found to be a potent, slow-binding inhibitor of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potency (KI*) of LeuSH was 7 nM while the corresponding alcohol l-leucinol (LeuOH) was a simple competitive inhibitor of much lower potency (KI=17 μM). These data suggest that the free thiol is likely involved in the formation of the E·I and E·I* complexes, presumably providing a metal ligand. In order to probe the nature of the interaction of LeuSH and LeuOH with the dinuclear active site of AAP, we have recorded both the electronic absorption and EPR spectra …

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The Methionyl Aminopeptidase From Escherichia Coli Can Function As An Iron(Ii) Enzyme, Ventris D'Souza, Richard Holz Mar 2015

The Methionyl Aminopeptidase From Escherichia Coli Can Function As An Iron(Ii) Enzyme, Ventris D'Souza, Richard Holz

Richard C. Holz

The identity of the physiologically relevant metal ions for the methionyl aminopeptidase (MetAP) from Escherichia coli was investigated and is suggested to be Fe(II). The metal content of whole cells in the absence and presence of expression of the type I MetAP from E. coli was determined by inductively coupled plasma (ICP) emission analysis. The observed change in whole cell concentrations of cobalt, cadmium, copper, nickel, strontium, titanium, and vanadium upon expression of MetAP was negligible. On the other hand, significant increases in the cellular metal ion concentrations of chromium, zinc, manganese, and iron were observed with the increase in …

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Exafs Studies Of Uteroferrin And Its Anion Complexes, Anne True, Robert Scarrow, Clayton Randall, Richard Holz, Lawrence Que Mar 2015

Exafs Studies Of Uteroferrin And Its Anion Complexes, Anne True, Robert Scarrow, Clayton Randall, Richard Holz, Lawrence Que

Richard C. Holz

Iron K-edge X-ray absorption data on the purple acid phosphatase from porcine uterus (uteroferrin, Uf) have been obtained for the native reduced enzyme and for the oxidized enzyme in its phosphate- and arsenate-bound forms. In all three complexes, the first sphere consists of 1.5 N/O at ∼1.94 Å, 4 N/O at ∼2.1 Å, and 0.5-1 N/O at ∼2.4 Å; in no complex is found an Fe-O bond of ∼1.8 Å which would derive from a μ-oxo bond. The ∼1.94-Å shell corresponds to Fe-OAr and Fe-μ-OH(or R) bonds. The ∼2.1-Å shell arises from histidine, carboxylate, oxoanion, and solvent ligation. The scatterer …

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Spectroscopic And Electrochemical Properties Of (Μ-Oxo)Diiron(Iii) Complexes Related To Diiron-Oxo Proteins. Structure Of [Fe2O(Tpa)2(Moo)4)](Clo4)2, Richard Holz, Timothy Elgren, Linda Pearce, Jian Zhang, Charles O'Connor, Lawrence Que Mar 2015

Spectroscopic And Electrochemical Properties Of (Μ-Oxo)Diiron(Iii) Complexes Related To Diiron-Oxo Proteins. Structure Of [Fe2O(Tpa)2(Moo)4)](Clo4)2, Richard Holz, Timothy Elgren, Linda Pearce, Jian Zhang, Charles O'Connor, Lawrence Que

Richard C. Holz

A series of (μ-oxo)diiron(III) complexes of tris(2-pyridylmethy1)amine (TPA), [Fe2O(TPA)2(L)] (C1O4)2, were synthesized and characterized where L represents the bridging tetraoxo anion ligands sulfate, phosphate, arsenate, vanadate, and molybdate. These tetraoxo anion complexes are the first (μ-oxo)diiron(III) complexes that reproduce the protein-tetraoxo anion stoichiometry found in purple acid phosphatases (PAPs). [Fe2O(TPA)2( MoO4)] (C104)2- CH3N (9) crystallizes in the monoclinic space group P21/n (a = 12.74(1) Å, b = 24.69(2) Å, c = 13.733(8) Å, β …

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Laser-Induced Europium(Iii) Luminescence And Nmr Spectroscopic Characterization Of Macrocyclic Diaza Crown Ether Complexes Containing Carboxylate Ligating Groups, Richard Holz, Scott Klakamp, C. Chang, William Horrocks Mar 2015

Laser-Induced Europium(Iii) Luminescence And Nmr Spectroscopic Characterization Of Macrocyclic Diaza Crown Ether Complexes Containing Carboxylate Ligating Groups, Richard Holz, Scott Klakamp, C. Chang, William Horrocks

Richard C. Holz

The Eu3+ and Y3+ complexes of 1,10-diaza-4,7,13,16-tetraoxacyclooctadecane-N, N' - diacetic acid (K22DA), 1,7-diaza-4,10,13-trioxacyclopentadecane-N, N' - diacetic acid (K21DA), and the open-chain analogue ethylene glycol bis (ß-aminoethyl ether) - N,-N,N',N'-tetraacetic acid (EGTA) have been characterized in solution by using Eu3+ laser-induced luminescence and 1H and 13CNMR spectroscopy. All of these ligands form 1:1 complexes with Eu3+ in solution with the luminescence lifetimes in H2O and D2O providing the number of coordinated water molecules. Stepwise changes in the coordination environment of the Eu3+ and Y3+ ions were monitored spectroscopically for each complex as a function of temperature. In addition, the Eu3+ spectral …

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Co-Catalytic Metallopeptidases As Pharmaceutical Targets, Richard Holz, Krzysztof Bzymek, Sabina Swierczek Mar 2015

Co-Catalytic Metallopeptidases As Pharmaceutical Targets, Richard Holz, Krzysztof Bzymek, Sabina Swierczek

Richard C. Holz

Understanding the reaction mechanism of co-catalytic metallopeptidases provides a starting point for the design and synthesis of new molecules that can be screened as potential pharmaceuticals. Many of the enzymes that contain co-catalytic metallo-active sites play important roles in cellular processes such as tissue repair, protein maturation, hormone level regulation, cell-cycle control and protein degradation. Therefore, these enzymes play central roles in several disease states including cancer, HIV, stroke, diabetes, bacterial infections, neurological processes, schizophrenia, seizure disorders, and amyotrophic lateral sclerosis. The mechanism of AAP, an aminopeptidase from Aeromonas proteolytica, is one of the best-characterized examples of a metallopeptidase containing …

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Characterization Of The Structural And Electronic Properties Of Spin-Coupled Dinuclear Copper(Ii) Centers By Proton Nmr Spectroscopy, Julie Brink, Rachel Rose, Richard Holz Mar 2015

Characterization Of The Structural And Electronic Properties Of Spin-Coupled Dinuclear Copper(Ii) Centers By Proton Nmr Spectroscopy, Julie Brink, Rachel Rose, Richard Holz

Richard C. Holz

The 1H NMR spectra of a series of well-characterized μ-phenoxo and μ-alkoxo spin-coupled dicopper(II) complexes have been investigated. The complexes studied were [Cu2(BPMP)(OH)]2+ (1) (BPMP = 2,6-bis[[bis(2-pyridylmethyl)amino]methyl]-4-methylphenol), [Cu2(CH3HXTA)(OH)]2- (2) (CH3HXTA = N,N ‘-(2-hydroxy-5-methyl-1,3-xylylene)bis(N-carboxymethylglycine), [Cu2(m-XYL)(OH)]2+ (3) (m-XYL = 2,6-bis[[bis(2-pyridylethyl)amino]methyl]phenol), and [Cu2(TBHP)(OAc)]2+ (4) (TBHP = N,N,N ‘,N  ‘-tetrakis[(2-benzimidazolyl)methyl]-2-hydroxy-1,3-diaminopropane). The magnetic interactions of these complexes range from strongly antiferromagnetically to weakly ferromagnetically coupled. Both one- and two-dimensional (COSY) 1H NMR methods were used to facilitate the assignment of the hyperfine shifted 1H NMR signals of each complex. COSY experiments provide clear cross signals for resonances <200 Hz wide. These data have facilitated the …

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The Catalytic Role Of Glutamate 151 In The Leucine Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Richard Holz Mar 2015

The Catalytic Role Of Glutamate 151 In The Leucine Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Richard Holz

Richard C. Holz

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% …

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X-Ray Crystallographic Characterization Of The Co(Ii)-Substituted Tris-Bound Form Of The Aminopeptidase From Aeromonas Proteolytica, Petra Munih, Aaron Moulin, Carin Stamper, Brian Bennett, Dagmar Ringe, Gregory Petsko, Richard Holz Mar 2015

X-Ray Crystallographic Characterization Of The Co(Ii)-Substituted Tris-Bound Form Of The Aminopeptidase From Aeromonas Proteolytica, Petra Munih, Aaron Moulin, Carin Stamper, Brian Bennett, Dagmar Ringe, Gregory Petsko, Richard Holz

Richard C. Holz

The X-ray crystal structure of the Co(II)-loaded form of the aminopeptidase from Aeromonas proteolytica ([CoCo(AAP)]) was solved to 2.2 Å resolution. [CoCo(AAP)] folds into an α/β globular domain with a twisted β-sheet hydrophobic core sandwiched between α-helices, identical to [ZnZn(AAP)]. Co(II) binding to AAP does not introduce any major conformational changes to the overall protein structure and the amino acid residues ligated to the dicobalt(II) cluster in [CoCo(AAP)] are the same as those in the native Zn(II)-loaded structure with only minor perturbations in bond lengths. The Co(II)–Co(II) distance is 3.3 Å. Tris(hydroxymethyl)aminomethane (Tris) coordinates to the dinuclear Co(II) active site …

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Kinetic And Spectroscopic Characterization Of The E134a- And E134d-Altered Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, Ryan Davis, David Bienvenue, Sabina Swierczek, Danuta Gilner, Lakshman Rajagopal, Brian Bennett, Richard Holz Mar 2015

Kinetic And Spectroscopic Characterization Of The E134a- And E134d-Altered Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, Ryan Davis, David Bienvenue, Sabina Swierczek, Danuta Gilner, Lakshman Rajagopal, Brian Bennett, Richard Holz

Richard C. Holz

Glutamate-134 (E134) is proposed to act as the general acid/base during the hydrolysis reaction catalyzed by the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae. To date, no direct evidence has been reported for the role of E134 during catalytic turnover by DapE. In order to elucidate the catalytic role of E134, altered DapE enzymes were prepared in which E134 was substituted with an alanine and an aspartate residue. The Michaelis constant (K m) does not change upon substitution with aspartate but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.09%), and alanine …

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Spectroscopic And Structural Characterization Of The Nine-Coordinate Adduct Of Tris(Dipivaloylmethanato)Europium(Iii) With 2,2':6',2"-Terpyridine, Richard Holz, L. Thompson Mar 2015

Spectroscopic And Structural Characterization Of The Nine-Coordinate Adduct Of Tris(Dipivaloylmethanato)Europium(Iii) With 2,2':6',2"-Terpyridine, Richard Holz, L. Thompson

Richard C. Holz

Tris(dipivaloylmethanato)(2,2':6',2"-terpyridine)europium(III), [Eu(DPM)3terpy], was synthesized from [EU(DPM)3] and 2,2':6',2"-terpyridine in carbon tetrachloride. The complex was characterized by infrared and luminescence spectroscopy. The infrared spectrum indicated all three nitrogen atoms of the terpyridine ligand were bound to the metal ion. The luminescence spectrum was recorded at 77 K and revealed a broad (15 cm-1) unresolvable single transition in the 5D0 -> 7F0 region. The 5D0 -> 7F1 region contained six bands and the 5D0 -> 7F2 region contained nine bands, indicating two distinct emitting metal centers. The luminescence spectrum suggested that the site symmetry of both metal centers is C1. The structure …

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Noesy Studies On The Fe(Iii)Co(Ii) Active Site Of The Purple Acid Phosphatase Uteroferrin, Richard Holz, Lawrence Que, Li-June Ming Mar 2015

Noesy Studies On The Fe(Iii)Co(Ii) Active Site Of The Purple Acid Phosphatase Uteroferrin, Richard Holz, Lawrence Que, Li-June Ming

Richard C. Holz

No abstract provided.

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The 1.20 Å Resolution Crystal Structure Of The Aminopeptidase From Aeromonas Proteolytica Complexed With Tris: A Tale Of Buffer Inhibition, William Desmarais, David Bienvenue, Krzysztof Bzymek, Richard Holz, Gregory Petsko, Dagmar Ringe Mar 2015

The 1.20 Å Resolution Crystal Structure Of The Aminopeptidase From Aeromonas Proteolytica Complexed With Tris: A Tale Of Buffer Inhibition, William Desmarais, David Bienvenue, Krzysztof Bzymek, Richard Holz, Gregory Petsko, Dagmar Ringe

Richard C. Holz

The aminopeptidase from Aeromonas proteolytica (AAP) is a bridged bimetallic enzyme that removes the N-terminal amino acid from a peptide chain. To fully understand the metal roles in the reaction pathway of AAP we have solved the 1.20 Å resolution crystal structure of native AAP (PDB ID = 1LOK). The high-quality electron density maps showed a single Tris molecule chelated to the active site Zn2+, alternate side chain conformations for some side chains, a sodium ion that mediates a crystal contact, a surface thiocyanate ion, and several potential hydrogen atoms. In addition, the high precision of the atomic …

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Spectroscopic Characterization Of A Series Of Europium(Iii) Amino Phosphonate Complexes In Solution, Richard Holz, Gretchen Meister, William Horrocks Mar 2015

Spectroscopic Characterization Of A Series Of Europium(Iii) Amino Phosphonate Complexes In Solution, Richard Holz, Gretchen Meister, William Horrocks

Richard C. Holz

The Eu3+ complexes of nitrilotris (methylenephosphonic acid) (ntp), ethylenediaminetetrakis (methylenephosphonic acid) (edtp), hexamethylenediaminetetrakis (methylenephosphonic acid) (hdtp), and diethylenetriaminepentakis (methylenephosphonic acid) (dtpp) have been characterized in solution by laser-induced Eu3+ luminescence spectroscopy. The latter three ligands form both 2:1 and 1:1 metal-ligand complexes, while ntp forms both 1:1 and 1:2 complexes at pH 6.0. The number of water molecules directly coordinating the metal ion was determined for each complex. The 7F0 -> 5D0 excitation spectra of edtp, hdtp, and dtpp reveal isomeric forms for both the 2:1 and 1:1 complexes while ntp exhibits only single species for both the 1:1 and …

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Structure Of 2,9-Dimethyl-1,10-Phenanthroline Hemihydrate, Doyle Britton, Larry Thompson, Richard Holz Mar 2015

Structure Of 2,9-Dimethyl-1,10-Phenanthroline Hemihydrate, Doyle Britton, Larry Thompson, Richard Holz

Richard C. Holz

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 > 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 …

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Kinetic And Spectroscopic Analysis Of The Catalytic Role Of H79 In The Methionine Aminopeptidase From Escherichia Coli, Sarah Watterson, Sanghamitra Mitra, Sabina Swierczek, Brian Bennett, Richard Holz Mar 2015

Kinetic And Spectroscopic Analysis Of The Catalytic Role Of H79 In The Methionine Aminopeptidase From Escherichia Coli, Sarah Watterson, Sanghamitra Mitra, Sabina Swierczek, Brian Bennett, Richard Holz

Richard C. Holz

To gain insight into the role of the strictly conserved histidine residue, H79, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H79A mutated enzyme was prepared. Co(II)-loaded H79A exhibits an overall >7000-fold decrease in specific activity. The almost complete loss of activity is primarily due to a >6000-fold decrease in kcat. Interestingly, the Km value obtained for Co(II)-loaded H79A was approximately half the value observed for wild-type (WT) EcMetAP-I. Consequently, kcat/Km values decreased only 3000-fold. On the other hand, the observed specific activity of Mn(II)-loaded …

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Kinetic And Structural Characterization Of Manganese(Ii)-Loaded Methionyl Aminopeptidases, Ventris D'Souza, Sabina Swierczek, Nathaniel Cosper, Lu Meng, Shane Ruebush, Alicja Copik, Robert Scott, Richard Holz Mar 2015

Kinetic And Structural Characterization Of Manganese(Ii)-Loaded Methionyl Aminopeptidases, Ventris D'Souza, Sabina Swierczek, Nathaniel Cosper, Lu Meng, Shane Ruebush, Alicja Copik, Robert Scott, Richard Holz

Richard C. Holz

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (Kd) for the first metal binding site were found to be 6 ± 0.5 and 1 ± 0.5 μM for EcMetAP-I and PfMetAP-II, respectively. These Kd values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 ± 0.2 and 1.4 ± 0.2 μM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 °C …

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Spectroscopic And X-Ray Crystallographic Characterization Of Bestatin Bound To The Aminopeptidase From Aeromonas (Vibrio) Proteolytica, Carin Stamper, David Bienvenue, Brian Bennett, Dagmar Ringe, Gregory Petsko, Richard Holz Mar 2015

Spectroscopic And X-Ray Crystallographic Characterization Of Bestatin Bound To The Aminopeptidase From Aeromonas (Vibrio) Proteolytica, Carin Stamper, David Bienvenue, Brian Bennett, Dagmar Ringe, Gregory Petsko, Richard Holz

Richard C. Holz

Binding of the competitive, slow-binding inhibitor bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoy]-leucine) to the aminopeptidase from Aeromonas proteolytica (AAP) was examined by both spectroscopic and crystallographic methods. Electronic absorption spectra of the catalytically competent [Co_(AAP)], [CoCo(AAP)], and [ZnCo(AAP)] enzymes recorded in the presence of bestatin revealed that both of the divalent metal ions in AAP are involved in binding bestatin. The electron paramagnetic resonance (EPR) spectrum of the [CoCo(AAP)]−bestatin complex exhibited no observable perpendicular- or parallel-mode signal. These data indicate that the two CoII ions in AAP are antiferromagnetically coupled yielding an S = 0 ground state and suggest that a single oxygen atom …

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Spectroscopically Distinct Geometrical Isomers In A Single Crystal. Characterization Of The Eight-Coordinate Adducts Of Tris(Dipivaloylmethanato)Lanthanide(Iii) With 2,9-Dimethyl-1,10-Phenanthroline, Richard Holz, Larry Thompson Mar 2015

Spectroscopically Distinct Geometrical Isomers In A Single Crystal. Characterization Of The Eight-Coordinate Adducts Of Tris(Dipivaloylmethanato)Lanthanide(Iii) With 2,9-Dimethyl-1,10-Phenanthroline, Richard Holz, Larry Thompson

Richard C. Holz

Tris(dipivaloylmethanato)(2,9-dimethyl- 1,10-phenanthroline)lanthanide(III), [L n(DPM)3*DMOP], complexes (Ln = La, Eu, Tb, Ho) were synthesized from [Ln(DPM)3] and 2,9-dimethyl- 1,10-phenanthroline in carbon tetrachloride. The Eu3+ and Tb3+ complexes were characterized spectroscopically by infrared and luminescence techniques. The luminescence spectra of the Eu3+ and Tb3+ adducts were recorded at 77 K. For Eu3+, two transitions in the 5Do →FO region are observed which are separated by an unprecedented 35 cm-l. The 5Do →7F1 and 5Do →7F2 spectral regions contain six and eight bands, respectively, indicating two distinct Eu3+ environments. The Eu3+ luminescence spectrum indicates that both Eu3+ environments have local site symmetry C1. …

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Synthesis, Molecular Structure And Reactivity Of A New Dicopper(Ii) Benzimidazole Complex With Non-Identical Copper(Ii) Sites, Richard Holz, Feben Gobena Mar 2015

Synthesis, Molecular Structure And Reactivity Of A New Dicopper(Ii) Benzimidazole Complex With Non-Identical Copper(Ii) Sites, Richard Holz, 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) has been synthesized in good yield by the condensation of 1,2-diaminobenzene with ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid. This ligand was used to synthesize the dinuclear CuII diacetonitrile complex [Cu2(EGTB-Et)(CH3CN)2](ClO4)4·2CH3CN (2). The X-ray structure shows two distinct square pyramidal CuII centres with each CuII ion bound by two benzimidazole nitrogen atoms, one amine nitrogen atom, an ether oxygen atom and one acetinitrile nitrogen atom. The Cu--Cu separation is 5.809A˚. Spectral titration of 2 with sodium acetate or sodium cyanide results in new complexes with terminal acetate or cyano groups. X-band EPR spectra of …

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Both Nucleophile And Substrate Bind To The Catalytic Fe(Ii)-Center In The Type-Ii Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja Copik, Sarah Waterson, Sabina Swierczek, Brian Bennett, Richard Holz Mar 2015

Both Nucleophile And Substrate Bind To The Catalytic Fe(Ii)-Center In The Type-Ii Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja Copik, Sarah Waterson, Sabina Swierczek, Brian Bennett, Richard Holz

Richard C. Holz

Metalloproteases utilize their active site divalent metal ions to generate a nucleophilic water/hydroxide. For methionine aminopeptidases (MetAPs), the exact location of this nucleophile, as well as of the substrate, with respect to the active site metal ion is unknown. In order to address this issue, we have examined the catalytically competent Fe(II)-loaded form of PfMetAP-II ([Fe(PfMetAP-II)]) in the absence and presence of both nitric oxide (NO) and the substrate-analogue inhibitor butaneboronic acid (BuBA) by kinetic and spectroscopic (EPR, UV−vis) methods. NO binds to [Fe(PfMetAP−II)] with a Kd of 200 μM forming an {FeNO}7 …

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Overexpression And Divalent Metal Binding Properties Of The Methionyl Aminopeptidase From Pyrococcus Furiosus, Lu Meng, Shane Ruebush, Ventris D'Souza, Alicja Copik, Susumu Tsunasawa, Richard Holz Mar 2015

Overexpression And Divalent Metal Binding Properties Of The Methionyl Aminopeptidase From Pyrococcus Furiosus, Lu Meng, Shane Ruebush, Ventris D'Souza, Alicja Copik, Susumu Tsunasawa, Richard Holz

Richard C. Holz

The gene encoding for the methionyl aminopeptidase from the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II; EC 3.4.11.18) has been inserted into a pET 27b(+) vector and overexpressed in Escherichia coli. The new expression system resulted in a 5-fold increase in purified enzyme obtained from a 5 L fermentor growth. The as-purified PfMetAP-II enzyme, to which no exogenous metal ions or EDTA was added, was found to have 1.2 equiv of zinc and 0.1 equiv of iron present by ICP-AES analysis. This enzyme had a specific activity of 5 units/mg, a 60-fold decrease from the fully loaded Fe(II) enzymes. When an additional …

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J-Dependent Curie Or Anti-Curie Behavior Of Proton Nmr Resonances For Antiferromagnetically Coupled Dinuclear Copper(Ii) Complexes, Richard Holz, Julie Brink, Rachel Rose Mar 2015

J-Dependent Curie Or Anti-Curie Behavior Of Proton Nmr Resonances For Antiferromagnetically Coupled Dinuclear Copper(Ii) Complexes, Richard Holz, Julie Brink, Rachel Rose

Richard C. Holz

No abstract provided.

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Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By L-Leucinephosphonic Acid. Spectroscopic And Crystallographic Characterization Of The Transition State Of Peptide Hydrolysis, Carin Stamper, Brian Bennett, Tanya Edwards, Richard Holz, Dagmar Ringe, Gregory Petsko Mar 2015

Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By L-Leucinephosphonic Acid. Spectroscopic And Crystallographic Characterization Of The Transition State Of Peptide Hydrolysis, Carin Stamper, Brian Bennett, Tanya Edwards, Richard Holz, Dagmar Ringe, Gregory Petsko

Richard C. Holz

The nature of the interaction of the transition-state analogue inhibitor l-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a Ki of 6.6 μM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of …

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Analyzing The Catalytic Mechanism Of The Fe-Type Nitrile Hydratase From Comamonas Testosteroni Ni1, Saroja Rao, Richard Holz Mar 2015

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 …

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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 Mar 2015

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 …

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