Alteration of the Substrate

The device most often used in the in vivo modification of the substrate is by the addition of unusual analogs of L-amino acids to the tissue culture medium. At moderate analog concentrations, viral protein synthesis continues, but the products contain the altered residues. This probably alters configuration of the precursors. 

Abnormally high temperatures are known to block viral protein cleavage with munerous viruses (l, 3 43) Usually, a few minutes at 39 41 C is sufficient to stop the cleavage reactions, which is a temperature range well below the point of heat inactivation of the proteases. Most likely the subtrates are being denatured, or reversibly altered, although effects on the host cells are possibly involved. 

Zinc ion is an inhibitor of viius production (44) and blocks protein cleavage of rhinovirus, enterovirus, and cardiovimis precursors (25). Compared to other methods of inhibition, zinc treatment is reasonably gentle, so that there is minimal cytotoxicity, and its effect is reversible by washing the treated cells in zinc- 

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There are significant differences in the control experiments that are possible in each of these systems. Before the quantifier bio- can be applied, the possibility of abiotic alteration of the substrate during incubation must be eliminated or taken into consideration. Only the first design lends itself readily to this control. For experiments using cell suspensions, the obvious controls are incubation of the substrate in the absence of cells or using autoclaved cultures. Care should be exercised in the interpretation of the results, however, since some reactions may apparently be catalyzed by cell components in purely chemical reactions. The question may then legitimately be raised whether or not these are biochemically mediated. Two examples are given as illustration of apparently chemically mediated reactions, which have been referred to in Chapter 1 ... 

Reeves, C.D., Murli, S., Ashley, G.W. et al. (2001) Alteration of the substrate specificity of a modular polyketide synthase acyltransferase domain through site-specific mutations. Biochemistry, 40, 15464. 

B. Lingen, D. Kolter-Jung, P. Dtinkelmann, R. Feldmann, J. Grotzinger, M. Pohl, M. Muller, Alteration of the substrate specificity of benzoylformate decarboxylase from Pseudomonas putida by directed evolution. ChemBioChem 2003, 4, 721-726. 

Regulation of enzymic activity occurs via two modes (cf. Ref. 50) alteration of the substrate binding process and/or alteration of the catalytic efficiency (turnover number) of the enzyme. The initial rate of a simple enzymatic reaction v is governed by the Michaelis-Menten equation... 

The adhesion to substrates may be increased by (i) mechanical alterations of the substrate, (ii) polar interactions with the bonding agent, or (iii) chemical bonding facilitated by an adhesion promoter [1], Although both mechanisms (i) and (ii) are effective in improving adhesion, this paper will focus on the use of chemical bonding (iii). 

Miller and Wolfenden6 compared the rates of decarboxylation of the substrate of orotidine-5 -monophosphate decarboxylase (OMPDC) in quantitative detail, on and off the enzyme. They showed that the apparent unimolecular rate constant of decarboxylation of the substrate when bound to the enzyme is about 1015 times greater than the decarboxylation process in the absence of the enzyme. Further studies confirm that the enzyme-promoted reaction does not involve additional intermediates or covalent alterations of the substrate. The reaction consists of carbon dioxide being formed and the resulting carbanion becoming protonated. Since thermodynamic barriers are not altered by catalysis, the energy of the carbanion must be a component that reflects the energy of the environment in which it is created, one in which the carbanion that is formed is selectively stabilized. 

A Schneider, T Stachelhaus, MA Marahiel. Targeted alteration of the substrate specificity of peptide synthetases by rational module swapping. Mol Gen Genet 57 308-318, 1998. 

Reeves CD, Murli S, Ashley GW, Piagentini M, Hutchinson CR, McDaniel R (2001) Alteration of the Substrate Specificity of a Modular Polyketide Synthase Acyltransferase Domain Through Site-Specific Mutations. Biochemistry 40 15464... 

Maves, S. A.,Yeom, H., Mclean, M. A., and Sligar, S. G., 1997, Decreased substrate affinity upon alteration of the substrate-docking region in cytochrome P450 BM3, FEBS Lett. 414 2136218. 

Such a moisture recovery translates into an increase in water activity (a ). However, as shown by Figure 7, the rate of most chemical and biochemical reactions increases as increases [4,18]. In consequence of these variations, phenomena such as drop in infectivity titers and/or alteration of the substrate may be observed during storage of freeze-dried products. 

There are significant differences in the control experiments that are possible in each of these systems. Before the quantifier bio- can be applied, the possibility of abiotic alteration of the substrate during incubation must be eliminated. On this point, only the first design lends itself readily to rigorous... 

Property modification techniques include alteration of the substrate by (i) doping (the process of intentionally introducing impurities into an extremely pure semiconductor substrate like silicon in order to change its electrical properties) and (ii) ion implantation (a technique by which ions of a material are implanted into another solid substrate, thereby changing the physical properties of the solid) [10]. 

The turbidimetric determination of hyaluronidase activity was introduced by Kass and Seastone (91). It may be considered as a refinement of the M. C. P. method, carried out with smaller amounts of purified substrate and an excess of acidified serum proteins at a rigidly controlled hydrogen ion concentration. (The ratio of hyaluronate to protein in the M. C. P. method of McClean is 1 5, in the method of Kass and Seastone it is 1 50, assuming a protein concentration in serum of 6%.) Under these conditions the substrate-protein precipitate forms a turbid suspension and can be measured in terms of its optical density. After incubation with suitable amounts of hyaluronidase, less substrate is available for the interaction with protein and the turbidity is reduced. Unlike the M. C. P. test which has as its end point the complete alteration of the substrate with respect to the interaction with acidified protein, the turbidimetric method is capable of measuring graded responses. 

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