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Articles 1 - 30 of 77
Full-Text Articles in Chemistry
Lysine Biosynthesis In Bacteria: A Metallodesuccinylase As A Potential Antimicrobial Target, Danuta M. Gillner, Daniel P. Becker Ph.D., Richard C. Holz
Lysine Biosynthesis In Bacteria: A Metallodesuccinylase As A Potential Antimicrobial Target, Danuta M. Gillner, Daniel P. Becker Ph.D., Richard C. Holz
Richard C. Holz
In this review, we summarize the recent literature on dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) enzymes, with an emphasis on structure–function studies that provide insight into the catalytic mechanism. Crystallographic data have also provided insight into residues that might be involved in substrate and hence inhibitor recognition and binding. These data have led to the design and synthesis of several new DapE inhibitors, which are described along with what is known about how inhibitors interact with the active site of DapE enzymes, including the efficacy of a moderately strong DapE inhibitor.
Identification Of A Histidine Metal Ligand In The Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Danuta M. Gillner, Sabina I. Swierczek, Dali Liu, Richard C. Holz
Identification Of A Histidine Metal Ligand In The Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Danuta M. Gillner, Sabina I. Swierczek, Dali Liu, Richard C. Holz
Richard C. Holz
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (Kd) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the …
The Dimerization Domain In Dape Enzymes Is Required For Catalysis, Boguslaw Nocek, Anna Starus, Magdalena Makowska-Grzyska, Blanca Gutierrez, Stephen Sanchez, Robert Jedrzejczak, Jamey C. Mack, Kenneth W. Olsen, Andrzej Joachimiak, Richard C. Holz
The Dimerization Domain In Dape Enzymes Is Required For Catalysis, Boguslaw Nocek, Anna Starus, Magdalena Makowska-Grzyska, Blanca Gutierrez, Stephen Sanchez, Robert Jedrzejczak, Jamey C. Mack, Kenneth W. Olsen, Andrzej Joachimiak, Richard C. Holz
Richard C. Holz
The emergence of antibiotic-resistant bacterial strains underscores the importance of identifying new drug targets and developing new antimicrobial compounds. Lysine and meso-diaminopimelic acid are essential for protein production and bacterial peptidoglycan cell wall remodeling and are synthesized in bacteria by enzymes encoded within dap operon. Thereforedap enzymes may serve as excellent targets for developing a new class of antimicrobial agents. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) convertsN-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. The enzyme is composed of catalytic and dimerization domains, and belongs to the M20 peptidase family. To understand the specific role …
Inhibitors Of Bacterial N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (Dape) And Demonstration Of In Vitro Antimicrobial Activity, Danuta Gillner, Nicola Armoush, Richard C. Holz, Daniel P. Becker
Inhibitors Of Bacterial N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (Dape) And Demonstration Of In Vitro Antimicrobial Activity, Danuta Gillner, Nicola Armoush, Richard C. Holz, Daniel P. Becker
Richard C. Holz
The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC50 = 3.3 μM, Ki = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.
Direct Patterning Of A Cyclotriveratrylene Derivative For Directed Self-Assembly Of C60, Zachary R. Osner, Dorjderem Nyamjav, Richard C. Holz, Daniel P. Becker
Direct Patterning Of A Cyclotriveratrylene Derivative For Directed Self-Assembly Of C60, Zachary R. Osner, Dorjderem Nyamjav, Richard C. Holz, Daniel P. Becker
Richard C. Holz
A novel apex-modified cyclotriveratrylene (CTV) derivative with an attached thiolane-containing lipoic acid linker was directly patterned onto gold substrates via dip-pen nanolithography (DPN). The addition of a dithiolane-containing linker to the apex of CTV provides a molecule that can adhere to a gold surface with its bowl-shaped cavity directed away from the surface, thereby providing a surface-bound CTV host that can be used for the directed assembly of guest molecules. Subsequent exposure of these CTV microarrays to C60 in toluene resulted in the directed assembly of predesigned, spatially controlled, high-density microarrays of C60. The molecular recognition capabilities of this CTV …
Identification Of A Histidine Metal Ligand In The Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Danuta Gillner, Sabina I. Swierczek, Dali Liu, Richard C. Holz
Identification Of A Histidine Metal Ligand In The Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade C. Mcgregor, Danuta Gillner, Sabina I. Swierczek, Dali Liu, Richard C. Holz
Richard C. Holz
The H355A, H355K, H80A, and H80K mutant enzymes of the argE-encoded N-acetyl-L-ornithine deacetylase (ArgE) from Escherichia coli were prepared, however, only the H355A enzyme was found to be soluble. Kinetic analysis of the Co(II)-loaded H355A exhibited activity levels that were 380-fold less than Co(II)-loaded WT ArgE. Electronic absorption spectra of Co(II)-loaded H355A-ArgE indicate that the bound Co(II) ion resides in a distorted, five-coordinate environment and Isothermal Titration Calorimetry (ITC) data for Zn(II) binding to the H355A enzyme provided a dissociation constant (Kd) of 39 μM. A three-dimensional homology model of ArgE was generated using the X-ray crystal structure of the …
The High-Resolution Structures Of The Neutral And The Low Ph Crystals Of Aminopeptidase From Aeromonas Proteolytica, William Desmarais, David Bienvenue, Krzysztof Bzymek, Gregory Petsko, Dagmar Ringe, Richard Holz
The High-Resolution Structures Of The Neutral And The Low Ph Crystals Of Aminopeptidase From Aeromonas Proteolytica, William Desmarais, David Bienvenue, Krzysztof Bzymek, Gregory Petsko, Dagmar Ringe, Richard Holz
Richard C. Holz
The aminopeptidase from Aeromonas proteolytica (AAP) contains two zinc ions in the active site and catalyzes the degradation of peptides. Herein we report the crystal structures of AAP at 0.95-Å resolution at neutral pH and at 1.24-Å resolution at low pH. The combination of these structures allowed the precise modeling of atomic positions, the identification of the metal bridging oxygen species, and insight into the physical properties of the metal ions. On the basis of these structures, a new putative catalytic mechanism is proposed for AAP that is likely relevant to all binuclear metalloproteases.
Acrylamide Production Using Encapsulated Nitrile Hydratase From Pseudonocardia Thermophila In A Sol–Gel Matrix, Salette Martinez, Misty Kuhn, James Russell, Richard Holz, Timothy Elgren
Acrylamide Production Using Encapsulated Nitrile Hydratase From Pseudonocardia Thermophila In A Sol–Gel Matrix, Salette Martinez, Misty Kuhn, James Russell, Richard Holz, Timothy Elgren
Richard C. Holz
The cobalt-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase) was successfully encapsulated in tetramethyl orthosilicate sol–gel matrices to produce a PtNHase:sol–gel biomaterial. The PtNHase:sol–gel biomaterial catalyzed the conversion of 600 mM acrylonitrile to acrylamide in 60 min at 35 °C with a yields of >90%. Treatment of the biomaterial with proteases confirmed that the catalytic activity is due to the encapsulated enzyme and not surface bound NHase. The biomaterial retained 50% of its activity after being used for a total of 13 consecutive reactions for the conversion of acrylonitrile to acrylamide. The thermostability and long-term storage of the PtNHase:sol–gel …
The Active Site Sulfenic Acid Ligand In Nitrile Hydratases Can Function As A Nucleophile, Salette Martinez, Rui Wu, Ruslan Sanishvili, Dali Liu, Richard C. Holz
The Active Site Sulfenic Acid Ligand In Nitrile Hydratases Can Function As A Nucleophile, Salette Martinez, Rui Wu, Ruslan Sanishvili, Dali Liu, Richard C. Holz
Richard C. Holz
Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp2) form to a tetrahedral form (sp3). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid …
A New Colorimetric Assay For Methionyl Aminopeptidases: Examination Of The Binding Of A New Class Of Pseudopeptide Analog Inhibitors, Sanghamitra Mitra, Anna Dygas-Holz, Jiri Jiracek, Miroslava Zertova, Lenka Zakova, Richard Holz
A New Colorimetric Assay For Methionyl Aminopeptidases: Examination Of The Binding Of A New Class Of Pseudopeptide Analog Inhibitors, Sanghamitra Mitra, Anna Dygas-Holz, Jiri Jiracek, Miroslava Zertova, Lenka Zakova, Richard Holz
Richard C. Holz
A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (l-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV–Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using l-Met-p-NA, the kinetic constants kcat and Km were determined for EcMetAP-I and PfMetAP-II …
Mechanistic Studies On The Aminopeptidase From Aeromonas Proteolytica: A Two-Metal Ion Mechanism For Peptide Hydrolysis, Guanjing Chen, Tanya Edwards, Ventris D'Souza, Richard Holz
Mechanistic Studies On The Aminopeptidase From Aeromonas Proteolytica: A Two-Metal Ion Mechanism For Peptide Hydrolysis, Guanjing Chen, Tanya Edwards, Ventris D'Souza, Richard Holz
Richard C. Holz
The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (Ki) of 30 mM. Thus, fluoride inactivates AAP only after substrate binding, and only a single fluoride ion binds to AAP. On the other hand, chloride ion does not inhibit AAP up to concentrations of 2 M at pH 8.0. The pH dependence of fluoride inhibition of AAP was measured over the pH range 6.0−9.5. Between pH values of 6.0 and 9.0, fluoride ion acts as a pure uncompetitive inhibitor of AAP, and the Ki increases from …
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Analyzing The Catalytic Role Of Asp97 In The Methionine Aminopeptidase From Escherichia Coli, Sanghamitra Mitra, Kathleen M. Job, Lu Meng, Brian Bennett, Richard C. Holz
Richard C. Holz
An active site aspartate residue, Asp97, in the methionine aminopeptidase (MetAPs) from Escherichia coli (EcMetAP-I) was mutated to alanine, glutamate, and asparagine. Asp97 is the lone carboxylate residue bound to the crystallographically determined second metal-binding site in EcMetAP-I. These mutant EcMetAP-I enzymes have been kinetically and spectroscopically characterized. Inductively coupled plasma–atomic emission spectroscopy analysis revealed that 1.0 ± 0.1 equivalents of cobalt were associated with each of the Asp97-mutated EcMetAP-Is. The effect on activity after altering Asp97 to alanine, glutamate or asparagine is, in general, due to a ∼ 9000-fold decrease in kca towards Met-Gly-Met-Met as compared to the wild-type …
Spectroscopic Characterization Of The Europium(Iii) Complexes Of A Series Of N,N'-Bis(Carboxymethyl) Macrocyclic Ether Bis(Lactones), Richard Holz, C. Chang, William Horrocks
Spectroscopic Characterization Of The Europium(Iii) Complexes Of A Series Of N,N'-Bis(Carboxymethyl) Macrocyclic Ether Bis(Lactones), Richard Holz, C. Chang, William Horrocks
Richard C. Holz
The Eu3+ and Y3+ complexes of a series of N,N'-bis(carboxymethyl) macrocyclic ether bis(lactone) ligands derived from ethyl-enediaminetetraacetic acid (EDTA) were characterized in solution by using Eu3+ laser-induced luminescence and 1H and 13CNMR spectroscopy. The complexation of EDTA was also studied for comparison purposes. These ligands form 1:1 complexes with Eu3+ at the concentrations studied (10 µM), with the luminescence lifetimes in H2O and D2O providing the number of coordinated water molecules. The 7F0 -> 5D0 excitation spectra indicate that for each of …
One- And Two-Dimensional Proton Nmr Studies Of The Active Site Of Iron(Ii) Superoxide Dismutase From Escherichia Coli, Li-June Ming, John Lynch, Richard Holz, Lawrence Que
One- And Two-Dimensional Proton Nmr Studies Of The Active Site Of Iron(Ii) Superoxide Dismutase From Escherichia Coli, Li-June Ming, John Lynch, Richard Holz, Lawrence Que
Richard C. Holz
The iron(II) superoxide dismutase (FeSOD) from Escherichia coli exhibits relatively sharp well-resolved paramagnetically shifted NMR signals. These signals can be associated with the endogenous ligands characterized by X-ray crystallography for the oxidized form FeIlISOD. Our results demonstrate that the active site remains intact upon reduction of the Fe(III) site, retaining the same coordination modes for the three N,-coordinated His residues (presumably His-26, His-75, and His-162) and the Asp residue (presumably Asp-158). The N-H resonances of the coordinated histidines are found at 88, 43, and 37 ppm, while the signals at 24.5 (E), 19 (G), and 15 (J) ppm …
Electrochemical Properties Of The Diiron Core Of Uteroferrin And Its Anion Complexes, Dan Wang, Richard Holz, Sheila David, Lawrence Que, Marian Stankovich
Electrochemical Properties Of The Diiron Core Of Uteroferrin And Its Anion Complexes, Dan Wang, Richard Holz, Sheila David, Lawrence Que, Marian Stankovich
Richard C. Holz
The reduction potentials (Em) of the purple acid phosphatase from porcine uterus, uteroferrin (Uf), and its phosphate, arsenate, and molybdate complexes were determined by coulometric methods at various pH values. The midpoint potential of Uf at the pH value for optimal enzyme activity (pH 5) was found to be +367 mV versus a normal hydrogen electrode (NHE), while at pH 6.01 Uf exhibits a reduction potential of +306 mV. At pH 6.01 molybdate was found to shift the potential of Uf more positive by 192 mV, while phosphate and arsenate shift the potential of Uf more negative by 193 and …
Mono-N-Acyl-2,6-Diaminopimelic Acid Derivatives: Analysis By Electromigration And Spectroscopic Methods And Examination Of Enzyme Inhibitory Activity, Jan Hlaváček, Miloslava Vítovcová, Petra Sázelová, Jan Pícha, Václav Vaněk, Miloš Buděšínský, Jiří Jiráček, Danuta Gillner, Richard Holz, Ivan Mikšík, Václav Kašička
Mono-N-Acyl-2,6-Diaminopimelic Acid Derivatives: Analysis By Electromigration And Spectroscopic Methods And Examination Of Enzyme Inhibitory Activity, Jan Hlaváček, Miloslava Vítovcová, Petra Sázelová, Jan Pícha, Václav Vaněk, Miloš Buděšínský, Jiří Jiráček, Danuta Gillner, Richard Holz, Ivan Mikšík, Václav Kašička
Richard C. Holz
Thirteen mono-N-acyl derivatives of 2,6-diaminopimelic acid (DAP)—new potential inhibitors of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE; EC 3.5.1.18)—were analyzed and characterized by infrared (IR) and nuclear magnetic resonance (NMR) spectroscopies and two capillary electromigration methods: capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Structural features of DAP derivatives were characterized by IR and NMR spectroscopies, whereas CZE and MEKC were applied to evaluate their purity and to investigate their electromigration properties. Effective electrophoretic mobilities of these compounds were determined by CZE in acidic and alkaline background electrolytes (BGEs) and by MEKC in acidic and alkaline BGEs containing a pseudostationary …
Kinetic And Spectroscopic Characterization Of The H178a Methionyl Aminopeptidase From Escherichia Coli, Alicja Copik, Sabina Swierczek, W. Lowther, Ventris D'Souza, Brian Matthews, Richard Holz
Kinetic And Spectroscopic Characterization Of The H178a Methionyl Aminopeptidase From Escherichia Coli, Alicja Copik, Sabina Swierczek, W. Lowther, Ventris D'Souza, Brian Matthews, Richard Holz
Richard C. Holz
To gain insight into the role of the strictly conserved histidine residue, H178, in the reaction mechanism of the methionyl aminopeptidase from Escherichia coli (EcMetAP-I), the H178A mutant enzyme was prepared. Metal-reconstituted H178A binds only one equivalent of Co(II) or Fe(II) tightly with affinities that are identical to the WT enzyme based on kinetic and isothermal titration calorimetry (ITC) data. Electronic absorption spectra of Co(II)-loaded H178A EcMetAP-I indicate that the active site divalent metal ion is pentacoordinate, identical to the WT enzyme. These data indicate that the metal binding site has not been affected by altering H178. …
The Aminopeptidase From Aeromonas Proteolytica: Structure And Mechanism Of Co-Catalytic Metal Centers Involved In Peptide Hydrolysis, Richard Holz
Richard C. Holz
Enzymes containing multi-metal active sites are central to numerous biological processes and, consequently, characterization of their structure and function is a problem of outstanding importance. One of the least-explored groups of enzymes is the hydrolases that contain dinuclear metal centers. These enzymes play key roles in carcinogenesis, tissue repair, and protein degradation processes. In addition, some of these enzymes can catalyze the hydrolysis of phosphorus(V) compounds found in nerve gases and agricultural neurotoxins. The determination of detailed reaction mechanisms for these enzymes is required for the design of highly potent, specific inhibitors that can function as potential pharmaceuticals. Hydrolytic enzymes …
Interaction Of Porcine Uterine Fluid Purple Acid Phosphatase With Vanadate And Vanadyl Cation, Debbie C. Carans, Carmen M. Simone, Richard C. Holz, Lawrence Que,Jr
Interaction Of Porcine Uterine Fluid Purple Acid Phosphatase With Vanadate And Vanadyl Cation, Debbie C. Carans, Carmen M. Simone, Richard C. Holz, Lawrence Que,Jr
Richard C. Holz
Uteroferrin, the purple acid phosphatase from porcine uterine fluid, is noncompetitively inhibited by vanadate in a time-dependent manner under both aerobic and anaerobic conditions. This time-dependent inhibition is observed only with the diiron enzyme and is absent when the FeZn enzyme is used. The observations are attributed to the sequential formation of two uteroferrin-vanadium complexes. The first complex forms rapidly and reversibly, while the second complex forms slowly and results in the production of catalytically inactive oxidized uteroferrin and V(IV), which is observed by EPR. The redox reaction can be reversed by treatment of the oxidized enzyme first with (V(1V)) …
The Aminopeptidase From Aeromonas Proteolytica Can Function As An Esterase, David Bienvenue, Rebecca Matthew, Dagmar Ringe, Richard Holz
The Aminopeptidase From Aeromonas Proteolytica Can Function As An Esterase, David Bienvenue, Rebecca Matthew, Dagmar Ringe, Richard Holz
Richard C. Holz
The aminopeptidase from Aeromonas proteolytica (AAP) can catalyze the hydrolysis of L-leucine ethyl ester (L-Leu-OEt) with a rate of 96±5 s–1 and a K m of 700 µM. The observed turnover number for L-Leu-OEt hydrolysis by AAP is similar to that observed for peptide hydrolysis, which is 67±5 s–1. The k cat values for the hydrolysis of L-Leu-OEt and L-leucine-p-nitroanilide (L-pNA) catalyzed by AAP were determined at different pH values under saturating substrate concentrations. Construction of an Arrhenius plot from the temperature dependence of AAP-catalyzed ester hydrolysis indicates that the rate-limiting step does not change as a function of temperature …
Characterization Of The Catalytically Active Mn(Ii)-Loaded Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade Mcgregor, Sabina Swierczek, Brian Bennett, Richard Holz
Characterization Of The Catalytically Active Mn(Ii)-Loaded Arge-Encoded N-Acetyl-L-Ornithine Deacetylase From Escherichia Coli, Wade Mcgregor, Sabina Swierczek, Brian Bennett, Richard Holz
Richard C. Holz
The catalytically competent Mn(II)-loaded form of the argE-encoded N-acetyl-l-ornithine deacetylase from Escherichia coli (ArgE) was characterized by kinetic, thermodynamic, and spectroscopic methods. Maximum N-acetyl-l-ornithine (NAO) hydrolytic activity was observed in the presence of one Mn(II) ion with k cat and K m values of 550 s−1 and 0.8 mM, respectively, providing a catalytic efficiency (k cat/K m) of 6.9 × 105 M−1 s−1. The ArgE dissociation constant (K d) for Mn(II) was determined to be 0.18 μM, correlating well with a value obtained by isothermal titration …
Structural Evidence That The Methionyl Aminopeptidase From Escherichia Coli Is A Mononuclear Metalloprotease, Nathaniel Cosper, Ventris D'Souza, Robert Scott, Richard Holz
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 …
Exafs Evidence For A "Cysteine Switch" In The Activation Of Prostromelysin, Richard Holz, Scott Salowe, Catherine Smith, Gregory Cuca, Lawrence Que
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 …
Methods For Fabricating Microarrays Of Motile Bacteria, Sergey Rozhok, Clifton Shen, Pey-Lih Littler, Zhifang Fan, Chang Liu, Chad Mirkin, Richard Holz
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 …
The Methionyl Aminopeptidase From Escherichia Coli Can Function As An Iron(Ii) Enzyme, Ventris D'Souza, Richard Holz
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 …
13C Nmr Analysis Of Biologically Produced Pyrene Residues By Mycobacterium Sp. Kms In The Presence Of Humic Acid, J. Karl C. Nieman, Richard C. Holz, Ronald C. Sims
13C Nmr Analysis Of Biologically Produced Pyrene Residues By Mycobacterium Sp. Kms In The Presence Of Humic Acid, J. Karl C. Nieman, Richard C. Holz, Ronald C. Sims
Richard C. Holz
Cultures of the pyrene degrading Mycobacterium sp. KMS were incubated with [4-13C]pyrene or [4,5,9,10-14C]pyrene with and without a soil humic acid standard to characterize the chemical nature of the produced residues and evaluate the potential for bonding reactions with humic acid. Cultures were subjected to a “humic acid/humin” separation at acidic pH, a duplicate separation followed by solvent extraction of the humic acid/humin fraction, and a high pH separation. 13C NMR analysis was conducted on the resulting solid extracts. Results indicated that the activity associated with solid extracts did not depend on pH and that approximately 10% of the added …
Exafs Studies Of Uteroferrin And Its Anion Complexes, Anne True, Robert Scarrow, Clayton Randall, Richard Holz, Lawrence Que
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 …
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
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 …
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
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 …
Co-Catalytic Metallopeptidases As Pharmaceutical Targets, Richard Holz, Krzysztof Bzymek, Sabina Swierczek
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 …