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- Zinc (7)
- Aminopeptidase (5)
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- Electron paramagnetic resonance (3)
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- Aeromonas proteolytica (2)
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- Aeromonas proteolytica (Vibrio proteolyticus) (1)
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Articles 1 - 30 of 77
Full-Text Articles in Physical Sciences and Mathematics
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.
Spectroscopically Distinct Cobalt(Ii) Sites In Heterodimetallic Forms Of The Aminopeptidase From Aeromonas Proteolytica: Characterization Of Substrate Binding, Brian Bennett, Richard Holz
Spectroscopically Distinct Cobalt(Ii) Sites In Heterodimetallic Forms Of The Aminopeptidase From Aeromonas Proteolytica: Characterization Of Substrate Binding, Brian Bennett, Richard Holz
Richard C. Holz
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 …
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 …
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 …
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 …
Epr And X-Ray Crystallographic Characterization Of The Product-Bound Form Of The MnIi-Loaded Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja Copik, Boguslaw Nocek, Sabina Swierczek, Shane Ruebush, Se Jang, Lu Meng, Ventris D'Souza, John Peters, Brian Bennett, Richard Holz
Epr And X-Ray Crystallographic Characterization Of The Product-Bound Form Of The MnIi-Loaded Methionyl Aminopeptidase From Pyrococcus Furiosus, Alicja Copik, Boguslaw Nocek, Sabina Swierczek, Shane Ruebush, Se Jang, Lu Meng, Ventris D'Souza, John Peters, Brian Bennett, Richard Holz
Richard C. Holz
Methionine aminopeptidases (MetAPs) are ubiquitous metallohydrolases that remove the N-terminal methionine from nascent polypeptide chains. Although various crystal structures of MetAP in the presence of inhibitors have been solved, the structural aspects of the product-bound step has received little attention. Both perpendicular- and parallel-mode electron paramagnetic resonance (EPR) spectra were recorded for the MnII-loaded forms of the type-I (Escherichia coli) and type-II (Pyrococcus furiosus) MetAPs in the presence of the reaction product l-methionine (l-Met). In general, similar EPR features were observed for both [MnMn(EcMetAP-I)]−l-Met and [MnMn(PfMetAP-II)]−l-Met. The observed perpendicular-mode EPR spectra consisted of a six-line hyperfine pattern at g = …
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 …
Kinetic, Spectroscopic, And X-Ray Crystallographic Characterization Of The Functional E151h Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Aaron Moulin, Sabina Swierczek, Dagmar Ringe, Gregory Petsko, Brian Bennett, Richard Holz
Kinetic, Spectroscopic, And X-Ray Crystallographic Characterization Of The Functional E151h Aminopeptidase From Aeromonas Proteolytica, Krzysztof Bzymek, Aaron Moulin, Sabina Swierczek, Dagmar Ringe, Gregory Petsko, Brian Bennett, Richard Holz
Richard C. Holz
Glutamate151 (E151) has been shown to be catalytically essential for the aminopeptidase from Vibrio proteolyticus (AAP). E151 acts as the general acid/base during the catalytic mechanism of peptide hydrolysis. However, a glutamate residue is not the only residue capable of functioning as a general acid/base during catalysis for dinuclear metallohydrolases. Recent crystallographic characterization of the d-aminopeptidase from Bacillus subtilis (DppA) revealed a histidine residue that resides in an identical position to E151 in AAP. Because the active-site ligands for DppA and AAP are identical, AAP has been used as a model enzyme to understand the mechanistic role of H115 in …
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 …
Spectroscopic And Thermodynamic Characterization Of The E151d And E151a Altered Leucine Aminopeptidases From Aeromonas Proteolytica, Krzysztof Bzymek, Sabina Swierczek, Brian Bennett, Richard Holz
Spectroscopic And Thermodynamic Characterization Of The E151d And E151a Altered Leucine Aminopeptidases From Aeromonas Proteolytica, Krzysztof Bzymek, Sabina Swierczek, Brian Bennett, Richard Holz
Richard C. Holz
Previous kinetic characterization of the glutamate 151 (E151)-substituted forms of the leucine aminopeptidase from Aeromonas proteolytica (Vibrio proteolyticus; AAP) has provided critical evidence that this residue functions as the general acid/base. The close proximity of similar glutamate residues to the bridging water/hydroxide of the dinuclear active sites of metalloenzymes (2.80 and 3.94 Å in carboxypeptidase G2 and 3.30 and 3.63 Å in AAP), suggests it may also be involved in stabilizing the active-site metal ions. Therefore, the structural perturbations of the dinuclear active site of AAP were examined for two E151-substituted forms, namely E151D-AAP and E151A-AAP, by …
Substrate Specificity, Metal Binding Properties, And Spectroscopic Characterization Of The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, David Bienvenue, Danuta Gilner, Ryan Davis, Brian Bennett, Richard Holz
Substrate Specificity, Metal Binding Properties, And Spectroscopic Characterization Of The Dape-Encoded N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase From Haemophilus Influenzae, David Bienvenue, Danuta Gilner, Ryan Davis, Brian Bennett, Richard Holz
Richard C. Holz
The catalytic and structural properties of divalent metal ion cofactor binding sites in the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. Co(II)-substituted DapE enzyme was 25% more active than the Zn(II)-loaded form of the enzyme. Interestingly, Mn(II) can activate DapE, but only to ∼20% of the Zn(II)-loaded enzyme. The order of the observed kcat values are Co(II) > Zn(II) > Cd(II) > Mn(II) >Ni(II) ∼ Cu(II) ∼ Mg(II). DapE was shown to only hydrolyze l,l-N-succinyl-diaminopimelic acid (l,l-SDAP) and was inactive toward d,l-, l,d-, and d,d-SDAP. DapE was also inactive toward several acetylated amino acids as …
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 …
Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Aliphatic Alcohols. Characterization Of The Hydrophobic Substrate Recognition Site, Leila Ustynyuk, Brian Bennett, Tanya Edwards, Richard Holz
Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Aliphatic Alcohols. Characterization Of The Hydrophobic Substrate Recognition Site, Leila Ustynyuk, Brian Bennett, Tanya Edwards, Richard Holz
Richard C. Holz
Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, …
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. …
Slow-Binding Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Peptide Thiols: Synthesis And Spectroscopic Characterization, Kristi Huntington, David Bienvenue, Yaoming Wei, Brian Bennett, Richard Holz, Dehua Pei
Slow-Binding Inhibition Of The Aminopeptidase From Aeromonas Proteolytica By Peptide Thiols: Synthesis And Spectroscopic Characterization, Kristi Huntington, David Bienvenue, Yaoming Wei, Brian Bennett, Richard Holz, Dehua Pei
Richard C. Holz
Peptide-derived thiols of the general structure N-mercaptoacyl-leucyl-p-nitroanilide (1a−c) were synthesized and found to be potent, slow-binding inhibitors of the aminopeptidase from Aeromonas proteolytica (AAP). The overall potencies (KI*) of these inhibitors against AAP range from 2.5 to 57 nM exceeding that of the natural product bestatin and approaching that of amastatin. The corresponding alcohols (2a−b) are simple competitive inhibitors of much lower potencies (KI = 23 and 360 μM). These data suggest that the free thiols are involved in the formation of the E·I and E·I* complexes, presumably serving as a metal ligand. To investigate the nature of the interaction …
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 …
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 …
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
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 …
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 …
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
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 …
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 …
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 …
Characterization Of The Structural And Electronic Properties Of Spin-Coupled Dinuclear Copper(Ii) Centers By Proton Nmr Spectroscopy, Julie Brink, Rachel Rose, Richard Holz
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 …