Nonenzymatic catalytic reactions

Catalytic reactions— Nonenzymatic Kinetic analysis through the use of catalyzed reactions is normally performed by the variable-time technique. This technique is appropriate because measured and constant quantities of A -H B in (21-23) can be used in a reaction. Typically, the time for completion of the reaction is measured and then related to the concentration of the catalyst. 

The earliest examples of analytical methods based on chemical kinetics, which date from the late nineteenth century, took advantage of the catalytic activity of enzymes. Typically, the enzyme was added to a solution containing a suitable substrate, and the reaction between the two was monitored for a fixed time. The enzyme s activity was determined by measuring the amount of substrate that had reacted. Enzymes also were used in procedures for the quantitative analysis of hydrogen peroxide and carbohydrates. The application of catalytic reactions continued in the first half of the twentieth century, and developments included the use of nonenzymatic catalysts, noncatalytic reactions, and differences in reaction rates when analyzing samples with several analytes. 

By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

Lin and coworkers disclosed that, at room temperature, nonenzymatic chemical addition was still observed in a water-organic solvent biphasic reaction system, though the volume of aqueous phases was relative small. Lin developed a method of preparing an active enzyme meal that contained essential water to retain its power for catalysis and found a new catalytic reaction system by application of the prepared meal in a nonaqueous monophasic organic medium (Figure 5.7). There was no problem over a wide range of temperature (from 0-30 °C) when the reactions were carried out under micro-aqueous conditions [50]. 

The interest in asymmetric synthesis that began at the end of the 1970s did not ignore the dihydroxylation reaction. The stoichiometric osmylation had always been more reliable than the catalytic version, and it was clear that this should be the appropriate starting point. Criegee had shown that amines, pyridine in particular, accelerated the rate of the stoichiometric dihydroxylation, so it was understandable that the first attempt at nonenzymatic asymmetric dihydroxylation was to utilize a chiral, enantiomerically pure pyridine and determine if this induced asymmetry in the diol. This principle was verified by Sharpless (Scheme 7).20 The pyridine 25, derived from menthol, induced ee s of 3-18% in the dihydroxylation of /rcms-stilbene (23). Nonetheless, the ee s were too low and clearly had to be improved. 

Those reactions of carboxylic acid and phosphoric acid derivatives which are susceptible to metal ion catalysis in nonenzymatic systems are almost without exception catalyzed by enzymes containing metal ions. This circumstantial evidence indicates strongly that the metal ions in the enzymatic reactions are concerned with the catalytic action, and not simply binding. 

In isomer 1, where catalytic redox functions are retained, a facilitated inactivation reaction such as oxazole formation, which can take place even nonenzymatically in any cell, results in potential toxicity. 

The catalytic strategy that an enzyme develops over evolutionary time is dictated by the chemistry of the reaction being catalyzed. The prolyl isomerases that have been studied to date are able to simply stabilize the nonenzymatic transition state without formation of covalent intermediates. Based on a value of lO" sec for CyP (Harrison and Stein, 1992 Kofron et ai, 1991) and a of sec" for the cis-to-trans isomerization of Suc-Ala-Ala-cis-Pro-Phe-pNA, we calculate an acceleration factor, of 10 , which corresponds to a transition state... 

A wide range of natural and unnatural monosaccharides has been generated by exploiting the catalytic capacity of aldolases which perform reactions equivalent to nonenzymatic aldol additions [54]. More than 20 aldolases have been identified so far and can be divided into three main groups, accepting either dihydroxyace-tone phosphate (DHAP), acetaldehyde, or pyruvic acid, and phosphoenolpyruvate as nucleophilic methylene component. A common feature is their high stereocontrol in the formation of the new C-C bond. As presented in Scheme 10 all four possible vicinal diols are accessible by selection of the appropriate DHAP-aldo-lase [2, 55], all of which show a distinct preference for the two stereocenters and a broad substrate tolerance for the aldehyde component. 

Because of their remarkable catalytic activity, a given number of enzyme molecules convert an enormous number of substrate molecules to products within a short time. Therefore, the appearance of increased amounts of enzymes in the blood stream is easily detected, although the amount of enzyme protein released from damaged cells is small compared with the total level of nonenzymatic proteins in bloods Thus a particular enzyme is recognized by its characteristic effect on a given chemical reaction despite the presence of a vast excess of other proteins. 

There has been considerable interest in the stereoselective ring opening of meso-cyclic anhydrides. The stereoselective alcoholysis of these anhydrides is particularly attractive as the resulting hemiesters are used as versatile intermediates in the construction of many bioactive compounds [1], Much effort has, therefore, been devoted to the development of efficient enzymatic and nonenzymatic catalytic systems for this reaction [2], Among the stereoselective catalysts developed to date,... 

An example of a quantitative SMR study correlating electronic properties and catalytic parameters is provided by the glutathione conjugation of para-substituted l-chloro-2-nitro-benzene derivatives (183). The values of log/j2 (second order rate constant of the nonenzy-matic reaction) and log (enzymatic reaction catalyzed by various glutathione transferase preparations) were correlated with the Hammett resonance cr value of the substrates, a measure of their electrophilicity. Regression equations with positive slopes and values in the range 0.88-0.98 were obtained. These results quantitate the influence of substrate electrophilicity on nucleophilic substitutions mediated by glutathione, be they enzymatic or nonenzymatic. 

Experiments reported by Pollack and his coworkers allow the conclusion that the dienolate anion intermediate is approximately isoenergetic with the more unstable unconjugated enone substrate/product, as proposed by Knowles and Albery in their theory for understanding optimization of catalytic efficiency [9]. Thus, based on the value of the rate constant for proton abstraction from the unconjugated enone, 1.7 x 10 s Pollack and coworkers calculated that the value of the Glint for proton abstraction from carbon is 10 kcal mol, a modest reduction from that expected ( 13 kcal mol ) for the nonenzymatic reaction. 

Examples for a high degree of promiscuity obviously include nonenzymatic proteins such as serum albumins that exhibit promiscuous catalytic activities (Table 1, entry 14). Other cases may include catalysis of unnatural reactions, meaning reactions for which, to our knowledge, no natural enzyme has evolved (e.g., the Kemp elimination performed by serum albumin (Table 1, entry 14, or siloxane hydrolysis by lipase (Table 1, entry 7)). 

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