Enzymes investigation

Desulfurization using purified enzymes Investigations into enzymatic desulfurization as an alternative to microbial desulfurization has revealed several enzymes capable of the initial oxidation of sulfur. A study reported use of laccase with azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as a mediator for oxidation of DBT [181]. The rate of this reaction was compared to hydrogen peroxide-based phosphotungstic acid-catalyzed oxidation and the latter was found to be about two orders of magnitude higher. The authors also oxidized diesel oil sulfur to no detectable levels via extraction of the oxidized sulfur compounds from diesel. In Table 9, the enzymes used in oxidation of DBT to DBTO are reported. 

The specificity of several of the enzymes identified in the 4S pathway of different organisms has been studied. In case of the DBT desulfurizing enzymes, little difference is expected in the specificity of the enzymes, say DszA, from different Rhodococcus strains found to date. This is essentially because the DNA sequence for the enzymes investigated so far has been the same. The difference in the specificity observed with whole cell assays is essentially due to the differences in substrate intake via the cell membrane and not necessarily due to a difference in the intrinsic enzyme specificity. It has been found that while isolated enzyme DszC (from KA2-5-1) can desulfurize up to 4,6 dipropyl DBT, whole cells cannot, indicating substrate transport as limiting factor. 

The use of zeolites to mirror biological systems is feasible in the two cases studied. Further work is in progress to extend the enzyme investigation. This is concerned with the replacement of Zn2+ by other metal ions, particularly those which are known to deactivate the enzyme. The work on skins will be extended to study vehicular transport of drugs in relation to known pharmaceutical preparations. 

Immobilized enzymes can be investigated in the feedback and in the GC mode. In the feedback mode oxidoreductases can be imaged. They use the SECM mediator as electron donor (or acceptor). Table 37.1 gives an overview about the enzymes investigated. 

An attempt was then made to obtain the homogeneous protein as a crystal for subsequent X-ray crystallography. After the 3D structure had been discerned, often the most laborious part of the whole enzyme investigation, the structure revealed the 3D coordination of the amino acids in folding. 

The study of //J-barrel proteins, also called TIM barrels after the first enzyme investigated in this class, triose phosphate isomerase, or (/3a)8-barrel enzymes after the... 

The first enzyme investigated by MCD was DMSOR where the Mo center is the sole chromophore (113). The Mo(V) state of DMSOR is formed only in substoichiometric amounts, making it impossible to study its features by electronic spectroscopy or MCD. An inactive, glycerol-inhibited form of the enzyme trapped in the Mo(V) state, which was robust under the required experimental conditions, was studied instead. The resultant MCD spectrum consisted of six transitions whose behavior (temperature dependence, magnetization) mapped precisely onto the expected six transitions of a dithiolene chelated to Mo. These results were duplicated for the tungsten enzyme AOR in Pfuriosus (114). However, note that the interpretation of these results was based on the (then) prevailing hypothesis that DMSOR was a mowomolybdopterin... 

Adedoyin A, Arns PA, Richards WO, et al. (1998) Selective effect of liver disease on the activities of specific metabolizing enzymes investigation of cytochromes P450 2C19 and 2D6. Clin Pharmacol Ther 64 8-17. 

Holocarboxylase synthetase from a wide variety of species wUl act on all four apocarboxylases from other species and on a variety of bacterial biotin-dependent apoenzymes. In all the biotin-dependent enzymes investigated to date, the reactive lysine residue is flanked by methionine residues on both sides, and there is a high degree of conservation of the amino sequence around this Met-Lys-Met sequence (Chapman-Smith and Cronan, 1999a, 1999b). 

Of potential interest are also the cellulolytic, xylanolytic and pollulan-hydrolyzing activities detected in Thermophilum strains and in AN 1, a member of the Thermococcales [59]. An additional candidate for biotechnical application is also represented by the a-glucosidase from P. furiosus, which exhibits the highest thermostability (to.5(98°C) = 46-48 h) and temperature optimum for activity (105-115 C) compared to all enzymes investigated so far [60] (Table 1). 

Fig. 7. (A) Flash-induced absorption-change pattern of neutral red in dark-adapted thylakoids in the absence and in the presence of hydroxylamine. (B) relative yield of proton release (measured by the magnitude of 4-ms rise component of 4A) in thylakoids in the presence of hydroxylamine solid dots, experimental values derived from (A) open circles for calculated values. See text for discussion. Figure source FOrster and Junge (1985) Interaction of hydroxylamine with the water-oxidizing enzyme investigated via proton release. Photochem Photobiol 41 192...

V F rster and W Junge (1985) Interaction of hydroxylamine with the water-oxidizing enzyme investigated via proton release. Photochem Photobiol 41 191-194... 

Whereas the mechanism of the C2-H deprotonation of ThDP has been shown to be identical in all ThDP-dependent enzymes investigated, the following steps in catalysis of the different enzymes require different protonation and deprotonation reactions of the intermediates formed along the process. In order to identify side chains involved in proton transfer steps, the distribution of reaction intermediates during catalysis of any wild type enzyme can be compared with that of active site mutant enzymes. Rate constants for single steps in catalysis can be calculated from... 

For all the enzymes investigated to date, inversion of configuration has always been observed for reactions known to be single-step processes. For reactions known to involve two steps (via the formation of a covalent enzyme-substrate intermediate), the stereochemical result is always retention. These results suggest that the general mechanism of enzyme-catalyzed phosphoryl transfer reactions always involves inversion, which rules out the adjacent pathway D but does not differentiate the dissociative pathway and the in-line associative pathway. 

Before the influence of freezing was studied, it was necessary to determine whether postmortem (p.m.) storage of muscle at temperatures above freezing (+4°C) influenced the subcellular distribution of the enzymes investigated. As is well known, storage of muscle p.m. results in drastic changes such as development of rigor mortis and a decline in pH from above 7 to about 5.5. 

The Hep-esters proved to be chemically stable during the removal of the N-terminal Z-, Boc- and the Aloe-group from the dipeptides 21. The selective removal of the Hep-esters was achieved by a lipase-catalyzed hydrolysis. From several enzymes investigated, a biocatalyst isolated from the fungus Rhizopus niveus was superior to the others with respect to substrate tolerance and reaction rate. The enzyme accepts a variety of Boc-, Z- and Aloc-protected dipeptide Hep-esters as substrates and hydrolyzes the ester functions in high yields at pH 7 and 37 °C... 

The diphosphate and triphosphate of nicotinamide have been prepared and their substrate properties for a number of polymerase enzymes investigated. The diphosphate is a good substrate for polynucleotide phosphorylase from... 

Steady-state techniques remain a major mode of enzyme investigation forming the basis of most enzyme assays, inhibition studies, pH profiles, etc. The detailed kinetics of this area are discussed well elsewhere in this volume [1] and our coverage here is selective of the common uses of steady-state kinetics. 

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