Molecular characteristics of the inhibition of human neutrophil elastase by nonsteroidal antiinflammatory drugs. (73/2113)

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.  (+info)

The zalpha domain of the editing enzyme dsRNA adenosine deaminase binds left-handed Z-RNA as well as Z-DNA. (74/2113)

The Zalpha domain of human double-stranded RNA adenosine deaminase 1 binds specifically to left-handed Z-DNA and stabilizes the Z-conformation. Here we report spectroscopic and analytical results that demonstrate that Zalpha can also stabilize the left-handed Z-conformation in double-stranded RNA. Zalpha induces a slow transition from the right-handed A-conformation to the Z-form in duplex r(CG)(6), with an activation energy of 38 kcal mol(-1). We conclude that Z-RNA as well as Z-DNA can be accommodated in the tailored binding site of Zalpha. The specific binding of Z-RNA by Zalpha may be involved in targeting double-stranded RNA adenosine deaminase 1 for a role in hypermutation of RNA viruses.  (+info)

Flavohemoglobin, a globin with a peroxidase-like catalytic site. (75/2113)

Biochemical studies of flavohemoglobin (Hmp) from Escherichia coli suggest that instead of aerobic oxygen delivery, a dioxygenase converts NO to NO3(-) and anaerobically, an NO reductase converts NO to N(2)O. To investigate the structural features underlying the chemical reactivity of Hmp, we have measured the resonance Raman spectra of the ligand-free ferric and ferrous protein and the CO derivatives of the ferrous protein. At neutral pH, the ferric protein has a five-coordinate high-spin heme, similar to peroxidases. In the ferrous protein, a strong iron-histidine stretching mode is present at 244 cm(-1). This frequency is much higher than that of any other globin discovered to date, although it is comparable to those of peroxidases, suggesting that the proximal histidine has imidazolate character. In the CO derivative, an open and a closed conformation were detected. The distal environment of the closed conformation is very polar, where the heme-bound CO strongly interacts with the B10 Tyr and/or the E7 Gln. These data demonstrate that the active site structure of Hmp is very similar to that of peroxidases and is tailored to perform oxygen chemistry.  (+info)

Redox properties and coordination structure of the heme in the co-sensing transcriptional activator CooA. (76/2113)

The CO-sensing transcriptional activator CooA contains a six-coordinate protoheme as a CO sensor. Cys(75) and His(77) are assigned to the fifth ligand of the ferric and ferrous hemes, respectively. In this study, we carried out alanine-scanning mutagenesis and EXAFS analyses to determine the coordination structure of the heme in CooA. Pro(2) is thought to be the sixth ligand of the ferric and ferrous hemes in CooA, which is consistent with the crystal structure of ferrous CooA (Lanzilotta, W. N., Schuller, D. J., Thorsteinsson, M. V., Kerby, R. L., Roberts, G. P., and Poulos, T. L. (2000) Nat. Struct. Biol. 7, 876-880). CooA exhibited anomalous redox chemistry, i.e. hysteresis was observed in electrochemical redox titrations in which the observed reduction and oxidation midpoint potentials were -320 mV and -260 mV, respectively. The redox-controlled ligand exchange of the heme between Cys(75) and His(77) is thought to cause the difference between the reduction and oxidation midpoint potentials.  (+info)

Vibrational spectroscopy of the oxygen-evolving complex and of manganese model compounds. (77/2113)

A number of molecularly specific models for the oxygen-evolving complex in photosystem II (PSII) and of manganese-substrate water intermediates that may occur in this process have been proposed recently. We summarize this work briefly. Fourier transform infrared techniques have emerged as fruitful tools to study the molecular structures of Y(Z) and the manganese complex. We discuss recent work in which mid-IR (1000-2000 cm(-1)) methods have been used in this effort. The low-frequency IR region (<1000 cm(-1)) has been more difficult to access for technical reasons, but good progress has been made in overcoming these obstacles. We update recent low-frequency work on PSII and then present a detailed summary of relevant manganese model compounds that will be of importance in understanding the emerging biological data.  (+info)

Haem-linked interactions in horseradish peroxidase revealed by spectroscopic analysis of the Phe-221-->Met mutant. (78/2113)

A gene encoding a Phe-221-to-Met substitution in the haem enzyme horseradish peroxidase has been constructed and expressed in Escherichia coli. In the wild-type enzyme the side chain of Phe-221 is tightly stacked against the imidazole ring of His-170, which provides the only axial ligand to the haem iron atom. The Phe-221-->Met enzyme is active, and forms characteristic complexes with typical peroxidase ligands (CO, cyanide, fluoride), and with benzhydroxamic acid. Significant differences between the mutant and wild-type enzymes can be detected spectroscopically. These include a change in the Fe(III) resting state of the enzyme to an unusual quantum mechanically mixed-spin haem species, a marked decrease in the pK(a) of the alkaline transition and a reduction in enzyme stability at alkaline pH for both Fe(III) and Fe(II) forms. The perturbation of the haem pocket in the mutant can be attributed to several factors, including the increased steric freedom and solvent accessibility of the His-170 ligand, as indicated by (1)H-NMR data, and the loss of the pi-pi interaction between His-170 and Phe-221.  (+info)

Integrity of thermus thermophilus cytochrome c552 synthesized by Escherichia coli cells expressing the host-specific cytochrome c maturation genes, ccmABCDEFGH: biochemical, spectral, and structural characterization of the recombinant protein. (79/2113)

We describe the design of Escherichia coli cells that synthesize a structurally perfect, recombinant cytochrome c from the Thermus thermophilus cytochrome c552 gene. Key features are (1) construction of a plasmid-borne, chimeric cycA gene encoding an Escherichia coli-compatible, N-terminal signal sequence (MetLysIleSerIleTyrAlaThrLeu AlaAlaLeuSerLeuAlaLeuProAlaGlyAla) followed by the amino acid sequence of mature Thermus cytochrome c552; and (2) coexpression of the chimeric cycA gene with plasmid-borne, host-specific cytochrome c maturation genes (ccmABCDEFGH). Approximately 1 mg of purified protein is obtained from 1 L of culture medium. The recombinant protein, cytochrome rsC552, and native cytochrome c552 have identical redox potentials and are equally active as electron transfer substrates toward cytochrome ba3, a Thermus heme-copper oxidase. Native and recombinant cytochromes c were compared and found to be identical using circular dichroism, optical absorption, resonance Raman, and 500 MHz 1H-NMR spectroscopies. The 1.7 A resolution X-ray crystallographic structure of the recombinant protein was determined and is indistinguishable from that reported for the native protein (Than, ME, Hof P, Huber R, Bourenkov GP, Bartunik HD, Buse G, Soulimane T, 1997, J Mol Biol 271:629-644). This approach may be generally useful for expression of alien cytochrome c genes in E. coli.  (+info)

In situ laser-Raman imagery of precambrian microscopic fossils. (80/2113)

Laser-Raman imagery is a sensitive, noninvasive, and nondestructive technique that can be used to correlate directly chemical composition with optically discernable morphology in ancient carbonaceous fossils. By affording means to investigate the molecular makeup of specimens ranging from megascopic to microscopic, it holds promise for providing insight into aspects of organic metamorphism and biochemical evolution, and for clarifying the nature of ancient minute fossil-like objects of putative but uncertain biogenicity.  (+info)