Studies of Specific Enzymes

Although aminoacyl-tRNA synthetases are necessary for protein synthesis in all tissues, their importance in chemical carcinogenesis is difficult to assess. Mutation induction by this pathway has been studied extensively (123), yet metabolic activation in a carcinogen-target tissue has not been demonstrated. The only exception is hepatic prolyl-tRNA synthetase activation of N-hydroxy-Trp-P-2 however, hepatic O-acetylation of this substrate also occurs to an appreciable extent (12). Further investigations involving the use of specific enzyme inhibitors would be helpful in addressing this problem. 

In an earlier spectrophotometric study of this enzyme, a red shift of the reduced nicotinamide absorbance had been noted in the difference spectrum of the binding of reduced coenzyme to the purified protein. Fisher et al.92> had pointed out that this is characteristic of most B-stereo-specific dehydrogenases, so Biellman et al. have made a successful prediction for Fisher. Fisher s suggestion that the supernatant and mitochondrial forms of malate dehydrogenase have different stereospecificities for NAD+ has not been substantiated, however 89>. 

Calu-3 cells have shown the ability to perform fatty acid esterification of budes-onide [132], In pre-clinical studies, this esterification results in a prolonged local tissue binding and efficacy, which is not found when the esterification is inhibited [133]. The precise mechanism remains undefined in that the identity of specific enzyme(s) responsible for this metabolic reaction is unclear [134], Assessment of the potential toxicity and metabolism of pharmaceuticals and other xenobiotics using in vitro respiratory models is still at its infancy. The development of robust in vitro human models (i.e., cell lines from human pulmonary origin) has the potential to contribute significantly to better understanding the role of biotransformation enzymes in the bioactivation/detoxication processes in the lung. 

The first studies of specificity were carried out using cholate, the glycine and taurine conjugates and taurine conjugates of the dihydroxy bile acids cheno-deoxycholate and ursodeoxycholate. Kramer and colleagues prepared plasma membrane vesicles from rat liver and compared bile-acid transport with values from CHO cells stably expressing NTCP. This work established that transport by the liver enzyme was maximal when 2 hydroxyls were present,... 

Reconstructed skin models may also be used to study photogenotoxicity. In fact, the comet assay was recently adapted to such models, using a specific technique, that is, dissociation and separation ofkeratinocytes after UV exposure of the reconstructed epidermis. Using a mixture of specific enzymes cocktail, it was possible to obtain suspension of cells without damaging them. For instance, the photocomet assay could be successfully performed for lomefioxacin after UVA exposure of reconstmcted epidermis [76], as shown in Figure 19.7. 

The information that there is a high probability that the metal lies in the carboxyl plane, but with possible deviations for specific metals, provides a mechanism for searching for metal-binding positions in proteins. This method was used in a study of the enzyme xylose isomerase from Streptomyces rubiginosus (Carrell et al., 1989). Two metal sites were located. One metal-binding site involves three carboxylates (aspartate and glutamate), histidine, and water, and the other involves four carboxylate... 

Although the metaphosphate mechanism for hydrolysis is well documented, such a pathway remains to be demonstrated in a biological system. Our present knowledge of many enzymic reactions allows, at best, the formulation of a preliminary mechanism, i.e. the chemical identity of substrates and enzymic intermediates and the minimal kinetic scheme. For example, much recent attention has been focused on the remarkable stability of the covalent phos-phoryl-enzyme (an O-phosphoryl serine) derived from E. coii alkaline phosphatase28 and inorganic phosphate, and on a systematic kinetic study of the enzyme s substrate specificity (O-, N- and S-monoesters) -9. Dephosphorylation of the enzyme, however, does not appear to be via a metaphosphate mechanism30. 

In this chapter we introduced some of the basic principles that govern protein structure. The discussion of protein structures begun in this chapter is continued in many other chapters in this text in which we consider structures designed for specific purposes. In chapter 5 we examine the protein structures for two systems the protein that transports oxygen in the blood and the proteins that constitute muscle tissue. In chapters 8 and 9 we discuss structures of specific enzymes. In chapters 17 and 24 we consider proteins that interact with membranes. In chapters 30 and 31 we study regulatory proteins that interact with specific sites on the DNA. And finally, in supplement 3 we examine the structures of immuno-globin molecules. 

By the use of specific enzymes, studies have been made in an attempt to classify the pressor substances in experimental hypertension. If a specific enzyme lowered blood pressure in hypertensive animals and not in normal ones, it was assumed that the substrate of that enzyme was attacked, and therefore contributed to the hypertension. Such assumptions, while not wholly valid, nevertheless pointed to certain substances as possibly concerned in hypertension. 

Mechanistic studies of RNA enzymes (ribozymes) and ribonucleoprotein (RNP) complexes such as the ribosome and telomerase, often seek to characterize RNA structural features, either dynamic or static, and relate these properties to specific catalytic functions. Many experimental techniques that probe RNA structure-function relationships rely upon site-specific incorporation of chemically modified ribonucleotides into the RNA of interest, often in the form of chemical cross-linkers to probe for sites of protein-RNA interaction or small organic fluorophores to measure dynamic structural properties of RNAs. The ability to arbitrarily modify any RNA molecule has been greatly enabled by modern RNA synthesis techniques however, there remains a practical size... 

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