Substrate independence

Many techniques ideally suited for nanostructure characterization unfortunately depend also on the substrate properties. For example, the reflectivity and conductivity of a substrate play an important role in the successful execution of the instrumental method. Hence, substrate-independent techniques are needed so that structure and/or behavior of the material can be investigated in a confined geometry, decoupled from the potentially invasive effect of the substrate-mate-rial interface. 

Fig. 3. Relationship between substrate lipophilicity (log P) and ability of screened microorganisms to metabolize model substrates (AP = Aminopyrine DZ = Diazepam TE = Testosterone TH = Theophylline WF = Warfarin). Log P is the logarithm of the partition coefficient of a compound between n-octanol and a pH 7.0 aqueous buffer. Ordinates represent the % of cultures tested able to transform the substrates, independently of the extent of metabolism and number of products formed (from [185])...

Aldol reactions have also been used as a means of macrocychzation in total synthesis and were quite successful in some cases. However, over a broader spectrum of substrates, the results are unpredictable at best and yields and stereochemical outcome vary greatly. The predominant reasons are difficulties in selective enolate formation in multi-carbonyl compounds, competing and equilibrating retro-aldolizations—especially with polyketides, which often possess several aldol moieties—and intermolecular instead of intramolecular reaction preference. Whereas most of these drawbacks may be overcome, substrate-independent stereocontrol plays a crucial role. At least one new stereocenter is formed during a macroaldolization, and because of the folding constraints involved, its configuration cannot be adequately predicted. Therefore, this can be useful in special cases but with the current possibilities is not the method of choice for a general diversity-oriented synthesis. 

Table 8). Evidently, stoichiometry and transport energetics are substrate-independent properties of the pump. The kinetics of the transport, however, is very much affected, by the substrate. This substrate specificity is not very well understood. It has been... 

The pKa at high pH is caused by a substrate-independent conformational change in the enzyme, a change that may be monitored directly by physical techniques such as optical rotation and fluorescence yield. Kinetic measurements give the same pKa as that found by these methods. 

Equation 12.15 shows that as far as kcJKM is concerned, it is as if a small fraction of the enzyme is permanently in the active conformation. This is the same for all substrates since K is substrate-independent. 

The rate of effusion can be predicted for any source with a defined geometry and for any material as a function of the source bottle temperature. Figure 2 shows a typical source bottle and a sketch showing how the initial charge behaves. The nozzle and orifice allow one to design for proper placement of the molecular species on the substrate independently of the design for the rate-limiting output. This aspect will be discussed quantitatively in the next section. 

If two different substrates bind simultaneously to the active site, then the standard Michaelis-Menten equations and competitive inhibition kinetics do not apply. Instead it is necessary to base the kinetic analyses on a more complex kinetic scheme. The scheme in Figure 6 is a simplified representation of a substrate and an effector binding to an enzyme, with the assumption that product release is fast. In Figure 6, S is the substrate and B is the effector molecule. Product can be formed from both the ES and ESB complexes. If the rates of product formation are slow relative to the binding equilibrium, we can consider each substrate independently (i.e., we do not include the formation of the effector metabolites from EB and ESB in the kinetic derivations). This results in the following relatively simple equation for the velocity ... 

B. Example 2 CYP3A Classification of Inhibitors Evaluation of Substrate Independence... 

This approach can be easily reproduced with other CYP3A-sensitive substrates, such as buspirone, triazolam, and eplerenone, and yields quantitative examination of the relationship between these sensitive substrates and midazolam. This question was pursued in some depth by Ragueneau-Majlessi et al. (22) and showed that classification of inhibitors (five moderate and eight potent) was substrate independent in 74% to 83% of the instances. Exceptions pertained to buspirone and simvastatin that seemed to be systematically more sensitive than midazolam and saquinavir that appeared less sensitive (22). Furthermore, results of this analysis allow an extrapolation of the inhibitory effect of a compound from one probe to another avoiding a duplication of studies (i.e., effects of inhibitor on simvastatin calculated from its effect on midazolam). 

By means of stopped-flow spectrophotometry, the reaction of flavopapain IV withN-benzyl-l,4-dihydronicotinamide (NBzNH) has been studied anaerobically. Using conditions of excess substrate and following the decrease in absorbance of Eox (at 427 nm) with time, we observed biphasic kinetic behavior. The experimental data could be fitted to a scheme using two consecutive first-order processes, and rate constants could be determined for the two phases. A reasonable interpretation of our results is that a labile intermediate is formed in the reaction, as illustrated in Equation 4, where ES represents the intermediate. In this interpretation, the second, substrate-independent, slower phase of the reaction corresponds to the breakdown of the intermediate ES (fc3 step in Equation 4). The initial, faster phase of the reaction corresponds to the formation of the intermediate from Eox and N-benzyl-l,4-dihydronicotinamide. Using Equation 5, the calculated rate constants for this phase, fcf, can be related to Ks and k2- Values of kCat and Km were calculated from the relationships of Equations 6 and 7, using the measured values of the kinetic parameters k2, 3, and Ks, and the numbers obtained were in reasonable agreement with the values obtained aerobically. 

The Menschutkin reaction of benzyl tosylates [21]-OTs with dimethyl-anilines or pyridines in acetonitrile generally proceed by a second-order bimolecular Sn2 mechanism for most ring-substituted compounds the plot of obs vs. [Nu] passes through the origin within experimental uncertainty (Yoh et al., 1989). However, for the reactions of strong ED derivatives under the same conditions, it was found that there was a significant intercept (i.e. a first-order component) in the kobs vs. [Nu] plots represented by (39) the intercept is a constant of the benzyl substrate independent of the amine nucleophiles, indicating a concurrent reaction zeroth-order in amine (Kim et al., 1995, 1998). 

White et al. (144) suggested that the biphasic deactivation of TD-i2 could be explained by an initial substrate-dependent fast phase that leads to a partially deactivated, conformationally altered enzyme (Ec). The conformation adopted would then allow dissociation of FMN in a substrate-independent manner leading to completely inactive enzyme (Ei) ... 

A remarkable feature of vicinal water is the observation that, to a first approximation, vicinal water occurs adjacent to most (or all) solid interfaces, regardless of the chemical nature of the surface of the solid and relatively independently of the nature and concentration of solutes in solution. This substrate independence has been termed the paradoxical effect for obvious reasons. 

Thermal anomalies in water near surfaces as diverse as diamond, glass, quartz, clays, mica, fatty acids, chondroitin 4-sulfate, polystyrene, polyvinyl acetate, cellulose, gelatin, and other biomacromolecules (such as enzymes and other proteins) in solution are known to occur close to the critical temperatures. Tj has also been shown to be unaffected by the concentration of electrolytes in solution. The concentrations of alkali chlorides (Li% Na", K, Rb and Cs" ) were varied by a factor of ICf with no detectable systematic effects on Tj (Drost-Hansen, 1985). However, the evidence for the paradoxical effect does not rely solely on the substrate independence of Tjj. 

Sun et al. (1986) have observed that various types of clay exhibit the same influence on the vicinal water. They concluded that a substrate independence exists. Packer (1977) also noted the substrate independence of water-structuring effects. He quotes Woessner s NMR studies showing that the ratio of the deuteron-to-proton splittings for water, oriented by proximity to a clay surface (3.75), appears independent of the type of clay and that the same ratio is found for water in oriented collagen, Li-DNA, and rayon. Thus, Packer suggests that it is merely the presence of a static surface and not its nature that matters in producing dynamic orientation of that water, and that the predominant effect is water-water interaction. The importance of the paradoxical effect lies in the prediction that vicinal water occurs at all solid interfaces and must, therefore, also occur in cellular systems—the cellular interior offers vast structural areas for the induction of vicinal water. 

---