Origins of Enzymes

A major experimental issue to be addressed is the rate and means by which particles are hydrolyzed and solubilized to provide substrates for heterotrophic bacteria, and the role of free enzymes in this process. Burns (1982) reviewed the possible locations and origin of enzyme activities in soils, and particularly underscored the potential importance of enzyme-humic complexes in microbial catalysis of substrates. As Burns (1982) discussed, enzymes associated with soil particles or humic substances are not subject to the same biochemical and physical restraints as are enzymes newly produced by microbial cells. Soil-held (or sediment-held) enzymes may therefore play a catalytic trigger role in substrate degradation, providing critical signals about substrate availability to the local microbial community. The conceptual model presented by Vetter et al. (1998) suggested that release of free enzymes into the environment may in fact represent... 

Stmctural analysis is often the basis for discussions of enzyme specihcity and mechanism. However, structural studies alone cannot establish mechanism or define the origins of enzyme specihcity. In the three examples cited above, structural studies were critical to illustrate the active site residues, but could not by themselves address the most pressing questions. In particular, enzyme specihcity is a kinetic phenomenon which is quantihed by kcatlKm, but the underlying origins of specihcity... 

Enzyme preparation Origin of enzyme Source of xylan Oligosaccharide structure M (degrees) References... 

This work addressed the issue of enzyme catalysis focusing on the principle of physical organic chemistry and the power of computer simulation approaches. It was shown that when such concepts as reorganization energy and Marcus parabolas are formulated in a consistent microscopic way, they could be used to explore the origin of enzyme catalysis. It was also clarified that phenomenological applications of the Marcus formula or related expressions can lead to problematic conclusions. 

In design of biosensors most often used are enzymes from oxidoreductases and hydrolases however, in very broad literature on this subject, applications of many other enzymes can be found. The analytical characteristics of enzymatic biosensor depend on numerous factors. Besides the origin of enzyme (type of natural material, from which the enzyme was isolated), the most significant is a mode of immobilization. It affects significantly kinetics of enzyme and diffusional limitations of the immobilization matrix. Although there is a general belief that enzymes should be immobilized in a hydrophilic environment, a successful immobilization of enzyme in carbon paste with silicon oil105 initiated development of numerous biosensors with enzyme... 

Before we proceed, it is important to clarify the procedure of parameter fitting, and its validation. The quantity to be compared in analyzing the origin of enzyme catalysis is the change in free energy of activation in the enzyme relative to an equivalent, uncatalyzed reaction in water. Therefore, it is reasonable to assume that the computational model that is parameterized for the aqueous reaction and can reproduce the reduction of free energy barrier in the enzyme can yield insights on enzyme catalysis. Nevertheless, we emphasize that it is equally critical to validate the computational model... 

In analyzing the origin of enzyme catalysis, Warshel and others have advocated the importance of comparing the enzymatic reaction with a reference reaction in water [32]. In addition, it is also necessary to study the reference reaction in the gas phase in order to understand the intrinsic reactivity and the effect of solvation. Thus, to understand enzyme catalysis fully, we must compare results for the same reaction in the gas phase (intrinsic reactivity), in aqueous solution (solvation effects), and in the enzyme (catalysis). This is not possible when there is no model reaction for the uncatalyzed process in the gas phase and in water, or if the uncatalyzed reaction is a bimolecular process as opposed to a unimolecular reaction in the enzyme active site. None of these problems apply to the ODCase reaction. Furthermore, OMP decarboxylation is a unimolecular process, both in water and the enzyme, providing an excellent opportunity to compare directly the computed free energies of activation [1] this is the approach that we have undertaken [16]. Warshel et al. used an ammonium ion-orotate ion pair fixed at distances of 2.8 or 3.5 A as the reference reaction in water to mimic an active site lysine residue [32]. 

It is clear that in a qualitative sense, the factors outlined in this section help us to understand the origin of enzyme catalytic power. However, it is not clear that for any given enzyme we can yet account quantitatively for the magnitude of its rate enhancement. 

Isoenzymes should become a valuable means of establishing the tissue origins of enzymes in serum. That this has not always been successfully accomplished stems very largely from the fact that isoenzyme forms are often not easy to separate. Sometimes, the required electrophoretic, chromatographic, or immunological procedures are not easily transplanted from their laboratories of origin. 

Figure 25.3 shows the relationship of active site of serine hydrolases. The serine hydrolases include serine proteases, lipases, and PHB depolymerases. A common feature of the serine proteases is the presence of a specific amino acid sequence -Gly-Xl-Ser-X2-Gly-. The catalytic mechanism of these enzymes is very similar and the catalytic center consists of a triad of serine, histidine, and aspartate residues [54]. The serine from this sequence attacks the ester bond nucleophilically [55]. Lipases and PHB depolymerases also have a common amino acid sequence around the active site, -Gly-Xl-Ser-X2-Gly-. These serine hydrolases may share a similar mechanism of substrate hydrolysis [21, 56]. In terms of origin of enzymes, it would be wise to consider that the enzyme had wide substrate specificity initially, and then it started to evolve gradually for each specific substrate. In the case of polyester hydrolysis, lipases showed the widest substrate specificity among serine hydrolases for hydrolysis of various polyesters ranging from a-ester bonds to (o-ester bonds. PHB depolymerases would become more specific for microbial PHB that has / -ester bonds, though it could also hydrolyze other polyesters that have -ester and y-ester bonds. Serine proteases such as proteinase K, subtilisin, a-chymotrypsin, elastase, and trypsin hydrolyze only optically active PLLA with a-ester bonds and various proteins with a-amido bonds. 

Mass spectrometry and chemometric methods cover very diverse fields Different origin of enzymes can be disclosed with LC-MS and multivariate analysis [45], Pyrolysis mass spectrometry and chemometrics have been applied for quality control of paints [46] and food analysis [47], Olive oils can be classified by analyzing volatile organic hydrocarbons (of benzene type) with headspace-mass spectrometry and CA as well as PC A [48], Differentiation and classification of wines can similarly be solved with headspace-mass spectrometry using unsupervised and supervised principal component analyses (SIMCA = soft independent modeling of class analogy) [49], Early prediction of wheat quality is possible using mass spectrometry and multivariate data analysis [50],... 

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