Article Information
PubMed
Related Articles
- The 1.4 Å Crystal Structure of KumamolysinThe 1.4 Å Crystal Structure of Kumamolysin
Structure, Volume 10, Issue 6, 1 June 2002, Pages 865-876
Mireia Comellas-Bigler, Pablo Fuentes-Prior, Klaus Maskos, Robert Huber, Hiroshi Oyama, Kenichi Uchida, Ben M. Dunn, Kohei Oda and Wolfram Bode
SummaryKumamolysin is a thermostable endopeptidase from novosp. MN-32, exhibiting maximal proteolytic activity around pH 3. It belongs to the newly identified family of serine-carboxyl proteinases, which also includes CLN2, a human lysosomal homolog recently implicated in a fatal neurodegenerative disease. Kumamolysin and its complexes with two aldehyde inhibitors were crystallized, and their three-dimensional structures were solved and refined with X-ray data to 1.4 Å resolution. As its homolog, kumamolysin exhibits a Ser/Glu/Asp catalytic triad with particularly short interconnecting hydrogen bonds and an oxyanion hole enabling the reactive serine to attack substrate peptide bonds at quite acidic pH. An additional Glu/Trp pair, unique to kumamolysin, might further facilitate proton delocalization during nucleophilic attack, in particular at high temperature.
Summary | Full Text | PDF (653 kb) - Electrorotation measurements of diamide-induced platelet act...Electrorotation measurements of diamide-induced platelet activation changes
Biophysical Journal, Volume 68, Issue 1, 1 January 1995, Pages 364-372
M. Egger and E. Donath
AbstractElectrorotation is a special dielectric spectroscopic technique capable of measuring the polarizability of single platelets. The rotational speed of the particles is recorded as a function of the frequency of the applied rotating electric field. As previously shown, the speed of electrorotation in the range of the first characteristic frequency (anti-field rotation) decreased upon activation and was correlated with [14C]serotonin release and an increase of the TMA-DPH-induced fluorescence. Diamide upon activation and was correlated with [14C]serotonin release and an increase of the TMA-DPH-induced fluorescence. Diamide incubation induced morphological changes in control platelets. These changes were accompanied by a shift of the first characteristic frequency of electrorotation toward higher values and a parallel increase of the anti-field rotation. This was explained by a decrease of membrane conductivity and by the changed polarizability of platelet interior due to the observed internal platelet structure changes. Diamide inhibited activation assessed by both electrorotation and TMA-DPH fluorescence in the case of all activators except the ionophore A 23187. Because diamide largely inhibited the A 23187-induced serotonin release, it was concluded that, despite the diamide treatment, the direct increase of cytoplasmic Ca2+ was still able to induce membrane conductivity changes accessible by electrorotation, but this did not complete the final release step of the activation process.
Abstract | PDF (1668 kb) - The ERO1Gene of Yeast Is Required for Oxidation of Protein D...The ERO1Gene of Yeast Is Required for Oxidation of Protein Dithiols in the Endoplasmic Reticulum
Molecular Cell, Volume 1, Issue 2, 1 January 1998, Pages 161-170
Alison R Frand and Chris A Kaiser
SummaryWe describe a conserved yeast gene, , that is induced by the unfolded protein response and encodes a novel glycoprotein required for oxidative protein folding in the ER. In a temperature-sensitive mutant, newly synthesized carboxypeptidase Y is retained in the ER and lacks disulfide bonds, as shown by thiol modification with AMS. apparently determines cellular oxidizing capacity since mutation of causes hypersensitivity to the reductant DTT, whereas overexpression of confers resistance to DTT. Moreover, the oxidant diamide can restore growth and secretion in mutants. Genetic tests distinguish the essential function of from that of . We show that glutathione is not required for CPY folding and conclude that Ero1p functions in a novel mechanism that sustains the ER oxidizing potential, supporting net formation of protein disulfide bonds.
Summary | Full Text | PDF (716 kb) - …more
Copyright © 1996 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 71, Issue 5, 2840-2847, 1 November 1996
doi:10.1016/S0006-3495(96)79480-8
Research Article
Aspartic proteinases: Fourier transform infrared spectroscopic studies of a model of the active side
G. Iliadis, B. Brzezinski and G. Zundel
Institute of Physical Chemistry, University of Munich, Germany.
Abstract
We synthesized and studied by Fourier transform infrared spectroscopy nine monosalts of diamides as models for the active side of aspartic proteinases. One compound, the monosalt of meta-aminobenzoic acid diamide of fumaric acid (m-FUM), shows the same biological activity as pepsin with regard to the splitting of peptide bonds of the Pro-Thi-Glu-Phe-Phe(4-NO2)-Arg-Leu heptapeptide. The monosalt of m-FUM forms with oxindole a complex in which the carboxylic acid group of the monosalt of m-FUM is strongly hydrogen bonded with the O atom of the peptide bond of oxindole. When one water molecule is added to this complex, the strong field of the carboxylate group destabilizes an O-H bond of the water molecule. The distorted water molecule attacks the carbon atom of the peptide group, and the water proton transfers to the peptide N atom. Simultaneously, the C-N bond of the amide group is broken. Hence it is demonstrated that the catalytic mechanism of aspartic acid proteinases is a base catalysis. The results show that for this catalytic mechanism there are sufficient carboxylic and carboxylate groups, as well as a water molecule in the correct arrangement. It was also demonstrated with other monosalts of dicarboxylic acids that well-defined steric conditions of the carboxylic acid and the carboxylate group must be fulfilled to show hydrolytic activity with regard to oxindole molecules.
