Enzymes reporter substrates

The possibility of isolating the components of the two above-reported coupled reactions offered a new analytical way to determine NADH, FMN, aldehydes, or oxygen. Methods based on NAD(P)H determination have been available for some time and NAD(H)-, NADP(H)-, NAD(P)-dependent enzymes and their substrates were measured by using bioluminescent assays. The high redox potential of the couple NAD+/NADH tended to limit the applications of dehydrogenases in coupled assay, as equilibrium does not favor NADH formation. Moreover, the various reagents are not all perfectly stable in all conditions. Examples of the enzymes and substrates determined by using the bacterial luciferase and the NAD(P)H FMN oxidoreductase, also coupled to other enzymes, are listed in Table 5. 

As discussed earlier, the tacit assumption of in vitro studies is that they are faithful reporters of how the enzymes and substrates will behave in vivo. At least qualitatively, the assumption seems largely to be true but quantitatively the assumption is less reliable. It assumes that the different microenvironments surrounding an enzyme in vivo and in an in vitro preparation do not differentially affect kinetic properties. It also assumes that, given equal concentrations of drug, the concentration that actually reaches the active site of the enzyme in the two different microenvironments will be equal (5). Clearly this does not need to be the case. As a consequence, a more reliable reporter of the in vivo kinetic properties of a drug would be highly desirable. 

Figure 8.11 Specific enzymatic immunodetection of a blotted protein. Depicted are blocked binding sites on the membrane (1), a primary antibody (2) specifically bound to an antigenic protein, and a secondary antibody (3) bound to the primary antibody. The secondary antibody is conjugated to a reporter enzyme (4). Substrate (S) is converted to insoluble product (P) at the site of the antigen.

The choice of organic solvent can also have a dramatic effect on selectivity.In contrast to enzyme activity, in the majority of examples reported there appears to be no correlation between solvent physical properties and enantioselectivity. In fact, investigating the effect of various solvents towards a number of lipases, Secundo et al also found that the optimal solvent differed with both enzyme and substrate. A number of theories have been postulated in order to explain these effects in individual cases, but none has any general predictive value. This is somewhat intriguing given that differences in enantioselectivity simply relate to a change in the relative rate of conversion of each enantiomer. 

The activity of [i-galactosidasc (P-Gal) was studied on a quartz chip using a static micromixer to mix the enzyme and substrate on the ms time scale. Inhibition by phenylethyl-P-D-thio-galactoside was also studied [1048]. In another report, the enzyme P-Gal was assayed on a chip in which P-Gal would convert a substrate, resoruhn-P-D-galactopyranoside (RBG), to resoruhn to be detected fluorescently [1049]. By varying the substrate concentrations and monitoring the amount of resoruhn by LIF, Michaelis-Menten constants could be determined. In addition, the inhibition constants of phenylethyl-P-D-thiogalactoside, lactose, and p-hydroxymercuribenzoic acid to the enzyme P-Gal were determined [1049]. 

Related systems were later developed for transaldolases 10-12 (Scheme 1.3) [12]. The fructo/tagato stereoselectivity of various transaldolases was determined by fluorescence for the stereoisomeric substrate pair 11/12. However, the reactivity of the substrates towards transaldolases is much lower than with the natural substrate due to the replacement of the phosphate group at position 6 of the natural fructose-6-phosphate substrate with the neutral, aromatic coumarin ether, which is not well recognized by the enzyme. Sevestre et al. [13] reported substrate 13 as a fluorogenic substrate for transketolases, based on a similar fluorescence release mechanism. 

The results of these studies and others reported previously demonstrate that the 1-oxypyridinyl group is an effective catalyst for the transacylation reactions of derivatives of carboxylic and phosphoric acids when incorporated in small molecules and polymers. Furthermore, this catalytic site exhibits high selectivity for acid chlorides in the presence of acid anhydrides, amides, and esters. Therefore, catalysts bearing this group as the catalytic site can be used successfully in synthetic applications that require such specificity. The results of this work suggest that functionalized polysiloxanes should be excellent candidates as catalysts for a wide variety of chemical reactions, because they combine the unique collection of chemical, physical, and dynamic-mechanical properties of siloxanes with the chemical properties of the functional group. Finally, functionalized siloxanes appear to mimic effectively enzyme-lipophilic substrate associations that contribute to the widely acknowledged selectivity and efficiency observed in enzymic catalysis. 

Metal-bearing enzymes such as leucine amino peptidase catalyze the hydrolysis of N-terminal peptide bonds through a process involving chelation between the enzyme, the substrate, and the metal ion. CoHman reported the selective N-terminal hydrolysis of simple peptides by cis-hydroxyaquotriethylenetetramine cobalt (III)... 

It was reported by Horecker and coworkers that one class of aldolases (called Class I to distinguish it from the Class II aldolase that is metal ion-dependent) could be inhibited by the addition of borohydride reducing agent to reaction mixtures containing both enzyme and substrate It was then shown for the fructose-1,6-bis-phosphate aldolase that the inhibition resulted from reduction of the Schiff base formed between the dihydroxyacetone phosphate substrate and the s-amino group of a lysine side chain, thereby compromising the ability of the lysine to participate in subsequent turnover. 

Typical commercial enzymes reported for resolution of amino acids were tested. Whole cell systems containing hydantoinase were found to produce only a-monosubstituted amino acids" the acylase-catalyzed resolution of Xacyl amino acids had extremely low rates toward a-dialkylated amino acids and the nitrilase system obtained from Novo Nordisk showed no activity toward the corresponding 2-amino-2-ethylhexanoic amide. Finally, a large-scale screening of hydrolytic enzymes for enantioselective hydrolysis of racemic amino esters was carried out. Of all the enzymes and microorganisms screened, pig hver esterase (PLE) and Humicola langinosa lipase (Lipase CE, Amano) were the only ones found to catalyze the hydrolysis of the substrate (Scheme 9.6). 

Kinetic and structural characterization of the known CoADR enzymes shows the occurrence of some variation with regard to their specificity. The S. aureus CoADR, a homodimer of 49kDa subunits, has a of 11 2 pmol 1 and 1.6 0.5 pmol for CoA disulfide and NADPH, respectively. Although an initial study of this enzyme reported ifcat values in the range of 1000 s (which would have had the enzyme operating near the limiting rate constant for a diffusion-controlled enzyme—substrate encounter), " subsequent kinetic analyses have shown the turnover number to be closer to 27 The enzyme shows 17% activity in the presence of... 

A more generally applicable method was reported in 2003 (referred to above as the TH method)," capable of trapping ThDP-bound intermediates shown in Schemes 1 and 2 by acid quench after rapid mixing of enzyme and substrate for predetermined time periods on a KINTEK chemical quench instrument. 

Enzymatic hydrolysis of polysaccharides (cellulose, starch) or oligosaccharides (maltose, saccharose, lactose) for the synthesis of food products is another class of processes MBR have been applied to. Paolucci-Jeanjean et al [4.56] have recently reported, for example, the production of low molecular weight hydrolysates from the reaction of cassava starch over a-amylase. In this case the UF membrane separates the enzyme and substrate from the reaction products for recycle. Good productivity without noticeable enzyme losses was obtained. Houng et al [4.57] had similar good success with maltose hydrolysis using the same type of MBR,... 

For T. viride cellulase substrate system a temperature of 50 °C. and a pH of 4.5-5.5 are reported (3) to be optimal. Over this pH range, loss of enzyme activity may be caused by adsorption on adsorbents such as Fullers earth or on substrates. Enzyme alone does not appear to lose activity at the temperature and pH of the reaction (50°C., pH 4.8-5.0) mixture. Rates of hydrolysis increase with enzyme and substrate con-... 

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