Enzymatic Systems

The conversion of cholesterol (2) to pregnenolone [145-13-1] (78) is accomplished primarily through enzymatic systems in the adrenocortical and gonadal mitochondria. This conversion appears to be rate-limiting and therefore is regarded as the control point for the entire steroid hormone... 

Germicidal Properties. The germicidal activity of aqueous chlorine is attributed primarily to HOCl. Although the detailed mechanism by which HOCl kills bacteria and other microorganisms has not been estabUshed, sufficient experimental evidence has been obtained to suggest strongly that the mode of action involves penetration of the cell wall followed by reaction with the enzymatic system. The efficiency of destmction is affected by temperature, time of contact, pH, and type and concentration of organisms (177). 

Figure 1 Schematic diagram depicting the partitioning of an enzymatic system into quantum and classical regions. The side chains of a tyrosine and valine are treated quantum mechanically, whereas the remainder of the enzyme and added solvent are treated with a classical force field.

The immobilization of enzymes with the formation of insoluble forms is usually intended for the development of specific catalysts for technical purposes. Here, we consider another medico-biological problem of the preparation of insoluble enzymatic systems based on crosslinked polyelectrolytes, used in the replacement therapy for oral administration. 

Sulphones occur relatively rarely as natural products. Thus there is no reported specific enzymatic system for the formation of sulphones. However, there are several well-understood pathways for the oxidation of sulphides to sulphoxides and these do lead to sulphones in some cases. 

No changes in GTP and y-GT activity were recorded after repeated administration of the above compounds. Also, histopathological examination did not point to liver necrosis. Similar phenomenon detected earlier after repeated administration of monobromobenzene, was interpreted as a result of damage of the microsomal enzymatic system responsible for the appearance of active metabolites (ref. 22). 

Sulfate reducers can use a wide range of terminal electron acceptors, and sulfate can be replaced by nitrate as a respiratory substrate. Molybdenum-containing enzymes have been discovered in SRB (also see later discussion) and, in particular, D. desulfuricans, grown in the presence of nitrate, generates a complex enzymatic system containing the following molybdenum enzymes (a) aldehyde oxidoreduc-tase (AOR), which reduces adehydes to carboxylic acids (b) formate dehydrogenase (FDH), which oxidizes formate to CO2 and (c) nitrate reductase (the first isolated from a SRB), which completes the enzy-... 

A bi-enzymatic system (glucose oxidase -I- HRP) was also used to catalyze the synthesis of phenolic polymers. The polymerization of phenol, albeit in moderate yield, was accomplished in the presence of glucose avoiding the addition of hydrogen peroxide (Scheme 2 ), which was formed in situ by the oxidation of glucose catalyzed by glucose oxidase. 

There have been many papers reporting studies on the influence of structure and conditions of the medium. Specifically, the kinetic behavior of enzymatic reactions in two-phase media was probed [7,25,27,63]. The reaction localization and the interaction between mass transfer and metabolism in compartmentalized media are interesting phenomena. Their study in the laboratory are useful for optimizing the operating conditions of bioreactors in a preparative scale. In addition, they also help to understand better the behavior of enzymatic systems in vivo. 

The approach presented here can help us better understand the behavior of some enzymatic systems in vivo. It can be useful for optimizing the operating conditions of bioreactors containing chemical species with amphiphilic properties. 

Methanotrophs rely on the enzymatic system methane monooxygenase (MMO) to catalyze the first step in the metabolism of methane, shown in Eq. (1) (1, 14). 

P. Mitchell (Nobel Prize for Chemistry, 1978) explained these facts by his chemiosmotic theory. This theory is based on the ordering of successive oxidation processes into reaction sequences called loops. Each loop consists of two basic processes, one of which is oriented in the direction away from the matrix surface of the internal membrane into the intracristal space and connected with the transfer of electrons together with protons. The second process is oriented in the opposite direction and is connected with the transfer of electrons alone. Figure 6.27 depicts the first Mitchell loop, whose first step involves reduction of NAD+ (the oxidized form of nicotinamide adenosine dinucleotide) by the carbonaceous substrate, SH2. In this process, two electrons and two protons are transferred from the matrix space. The protons are accumulated in the intracristal space, while electrons are transferred in the opposite direction by the reduction of the oxidized form of the Fe-S protein. This reduces a further component of the electron transport chain on the matrix side of the membrane and the process is repeated. The final process is the reduction of molecular oxygen with the reduced form of cytochrome oxidase. It would appear that this reaction sequence includes not only loops but also a proton pump, i.e. an enzymatic system that can employ the energy of the redox step in the electron transfer chain for translocation of protons from the matrix space into the intracristal space. 

The understanding of the catalytic function of enzymes is a prime objective in biomolecular science. In the last decade, significant developments in computational approaches have made quantum chemistry a powerful tool for the study of enzymatic mechanisms. In all applications of quantum chemistry to proteins, a key concept is the active site, i.e. a local region where the chemical reactivity takes place. The concept of the active site makes it possible to scale down large enzymatic systems to models small enough to be handled by accurate quantum chemistry methods. 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

The next section contains the most relevant findings from ONIOM applications to enzymatic systems performed in our group. This is followed by a discussion of the important protein effects and how this information can be used to improve the modeling of enzymatic reactions. 

Theoretical studies that has investigated the homolysis step in different enzymatic systems [68-70] reveal that small models comprising only the corrin ring and two ligands are insufficient and that inclusion of more amino acids are essential to stabilize the radical intermediates. Recently, a QM/MM study of the initial phase of the glutamate mutase-catalyzed reaction found a large electrostatic stabilization by the surrounding protein [70], In our study of MCM we employed the ONIOM QM MM approach to reveal the role of the protein in the rupture of the Co—C5 bond [29],... 

Zinc hydroxide and alkoxide species are particularly relevant to catalytic processes, often forming the active species. The cooperative effects of more than one zinc ion and bridged hydroxides are exploited in some enzymatic systems. Zinc alkyl phosphate and carboxylate materials have been important in the formation of framework compounds, often containing large amounts of free space for the inclusion of guest molecules. Aldehyde and ketone compounds are of low stability due to the poor donor capabilities of the ligands however, a number of examples have recently been characterized. 

Theoretical calculations have been carried out on a number of zinc-containing enzymatic systems. For example, calculations on the mechanism of the Cu/Zn enzyme show the importance of the full protein environment to get an accurate description of the copper redox process, i.e., including the electronic effects of the zinc ion.989 Transition structures at the active site of carbonic anhydrase have been the subject of ab initio calculations, in particular [ZnOHC02]+, [ZnHC03H20]+, and [Zn(NH3)3HC03]+.990... 

Such an inter-type difference will not be utilized in this book, mainly because it complicates the classification and is not necessary as tbe focus is placed on the substrates and the products. The argument is also valid for enzymatic transformations [12d, 14], where one enzymatic system with one enzyme or different independent enzymatic systems with one or more enzymes may be used. In Nature, as well as in several artificial enzymatic domino reactions, a mixture of different enzymes catalyzing independent cycles is employed. 

In the processes that require regeneration of cofactors such as nicotinamide adenine dinucleotide phosphate (NAD(P)H) and adenosine triphosphate (ATP), whole-cell biotransformations are more advantageous than enzymatic systems [12,15]. Whole cells also have a competitive edge over the isolated enzymes in complex conversions involving multiple enzymatic reactions [14]. 

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