Active site studies

To understand the effect of the protein on this modeled reaction mechanism, we selected the first reaction step, H2O2 reduction by a glutathione molecule for further investigations using the ONIOM (QM MM) method [28], The computational setup was similar to the structural study, but the effects of the additional water molecules were added from the active-site model. It is assumed that the reaction coordinate is the same as in the active-site study and no additional reaction pathways were investigated. An important point of the present ONIOM study is the full optimization of QM MM transition states using the novel ONIOM algorithms [9],... 

J. Eyzaguirre, ed., Chemical modification of enzymes, active site studies, Ellis Norwood series in biochemistry, Ellis Horwood Ltd. (1987). 

IF Aparicio, P Caffrey, AFA Marsden, I Staunton, PF Leadlay. Limited proteolysis and active site studies of the first multienzyme component of the erythromycin-producing polyketide synthase. I Biol Chem 269 8524-8528, 1994. 

Eyzaguirre, J. (Ed.) (1987) Chemical Modification of Enzymes Active Site Studies. Ellis Horwood,... 

P9. Plotch, S., and Lukton, A., Active site studies of alkaline phosphatase. Biochim. Biophys. Acta 99, 181-182 (1965). 

Immobilization of cholinesterase, like that of other enzymes, has a number of potentially valuable applications, including active-site studies, economical use of the enzyme as an analytical reagent, and enhancement of the storage life of the enzyme. 

Galactose-l-P uridylyltransferase from Escherichia coli has been purified and its molecular properties characterized (29). It was found to have a M, of 80,(X)0 and to contain two subunits with a Mr of about 40,000. Active site studies showed the subunits to be functionally identical, so that it could be assumed that they are structurally identical (30). Nucleotide sequence analysis of the cloned gene gaU... 

Molecular weight measurements are far less sensitive to variables which are by their nature most affected by experimental error, such as catalyst amount or catalyst/monomer purity, and, more important, on the number of active centers, but are highly sensitive to the catalyst structure, monomer concentration, and polymerization temperature. Hence, reliable molecular weights can give much information on the nature of the active sites. Studies of this kind have been reported by Miilhaupt and Brintzinger on catalysts C2-T24 and 25, by Kaminsky and Werner on three Ci-symmetric zirconocenes, and... 

Schirmer A, Jendrossek D, ScUegel HG (1993) Degradation of poly(3-hydroxyoctanoic add) [P(3HO)] by bacteria purification tmd properties of a P(3HO) depolymerase fmm Pseudomonas fluorescens GK13. Appl Environ Microbiol 59 1220 Schirmer A, Matz C, Jendrossek D (1995) Substrate spedfidties of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic add) depolymerase of Pseudmnonas fluorescens GK13. Can J Microbiol 41 170-179 Schmid A, KoUmer A, Sonnleitner B, Witholt B (1999) Development of equipment and procedures for the safe operation of aerobic bacterid bioprocesses in the presence of bulk amounts of flammable oiganic solvents. Bioprocess Eng 20 91-100 Schmid A, Dordick JS, Hauer B, Kiener A, Wubbolts M, Witholt B (2001) Industrial biocatalysis today and tomorrow. Nature 409 258-268... 

Brucato CL, Wong SS (1991) Extracellular poly(3-hydroxybutyrate) depolymerase from PeniciUium funiculosum general characteristics and active site studies. Arch Biochem Biophys 290 497-502... 

Another interesting version of XAS suitable for active site studies is soft X-Ray absorption spectroscopy. The L edges of first-row transition metals often bear a considerable diagnostic potential for the identification of oxidation states and coordinations, which at present is largely disregarded in catalysis research. 

Nevertheless, solid-state NMR can be usefiil for active site studies, which is nowadays predominantly employed for acid catalysis, e.g., with zeolites [32]. Chemical shifts obtained are related to the electronic state of the atom H-NMR data have been, for instance, used to determine acidity of Brpnsted sites in zeolites, NMR can discriminate in various environments in supported catalysts [33]. As long as the sites are sufficiently dilute, NMR intensity is directly related to concentration. Structural information is accessible by echo techniques which allow determining distances between spin sites. By analogy with IR methodology, adsorption sites have been investigated with probe molecules also in NMR studies. While work with N bases may require isotopic enrichment of the probe, there are opportunities to detect subtle structural features by cross-polarization and doubleresonance (e.g., " N/ A1) techniques [34]. 

The in-situ application of solid-state NMR requires heating and temperature control of the spinning sample. Techniques available for this purpose are reviewed in [35]. The method is applied preferentially for mechanistic rather than for active site studies, but the former often result in indirect conclusions on the sites involved in the detected reaction mechanisms [32]. 

The methods discussed so far are particularly qualified for active site studies by offering the potential for in situ work—even in electron microscopy, an environmental version in which the sample is kept under some millibar of reactant pressure is available nowadays. Valuable insight into the structure of surfaces can be, however, also obtained by methods targeting adsorptive interactions of reactants or other probe molecules with the surface although these are usually performed in separate experiments, e.g., on catalysts previously subjected to reaction conditions. 

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