Hydrolysis rate, enzyme-increased

Observed rates of hydrolysis vary enormously depending upon the structure of the ester and the experimental conditions used (Chapter 5.6). In addition, the intervention of enzymes in biosystems can increase hydrolysis rates by as much as 10 . 

The activation barrier for the hydrolysis of sucrose into glucose and fructose is 108 kJ/mol. If an enzyme increases the rate of the hydrolysis reaction by a factor of 1 million, how much lower must the activation barrier be when sucrose is in the active site of the enzyme (Assume that the frequency factors for the catalyzed and uncatalyzed reactions are identical and a temperature of 25 °C.)... 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

Caramel color interacts with other food components. As an example, a concentration higher than 700 ppm caramel in cola increased the rate of hydrolysis of the aspartame, forming alpha-L-aspartyl-L-phenylalanine. Caramelization products inhibited enzymic browning by 85.8 and 72.2% when heated at pH 4 and 6, respectively, for 90 min. The highest inhibitory activity was found for the fraction with molecular weight of 1000 to 3000. Caramel is often used for adulteration of juices and other foods like honey or coffee. It can be determined by quantification of marker molecules such as 5-HMF, 4-Mel, and DFAs. ... 

In some cases enzymes can increase the rate of reaction by up to lO times. Carnell and Roberts (1997) have briefly discussed the scope of biotransformations that are used to make pharmaceuticals like penicillins, cephalosporines, erythromycin, lovastatin, cyclosporin, etc., and for food additives like citric acid, L-glutamate, and L-lysine. A very successful transformation by Zeneca has been that of benzene reduction, with Pseudomonase Putida, to dihydrocatechol and catechol the dihydro derivative is used to produce (+/-) pinitol. Fluorobenzene has been converted to fluorodihydrocatechol, an intermediate for pharmaceuticals. The highly stereo selective Bayer-Villeger reaction has been carried out with genetically engineered S-cerevisvae. Hydrolases have allowed enantioselective, and in some cases regioselective, hydrolysis of racemic esters. 

Unfortunately, the size of the crystallographic problem presented by elastase coupled with the relatively short lifedme of the acyl-enzyme indicated that higher resolution X-ray data would be difficult to obtain without use of much lower temperatures or multidetector techniques to increase the rate of data acquisition. However, it was observed that the acyl-enzyme stability was a consequence of the known kinetic parameters for elastase action on ester substrates. Hydrolysis of esters by the enzyme involves both the formation and breakdown of the covalent intermediate, and even in alcohol-water mixtures at subzero temperatures the rate-limidng step is deacylation. It is this step which is most seriously affected by temperature, allowing the acyl-enzyme to accumulate relatively rapidly at — 55°C but to break down very slowly. Amide substrates display different kinetic behavior the slow step is acylation itself. It was predicted that use of a />-nitrophenyl amid substrate would give the structure of the pre-acyl-enzyme Michaelis complex or even the putadve tetrahedral intermediate (Alber et ai, 1976), but this experiment has not yet been carried out. Instead, over the following 7 years, attention shifted to the smaller enzyme bovine pancreatic ribonuclease A. 

Gin > Val > Ser > Trp, Pro, Glu, Phe, Nph, Arg, while for HNC the order is Tyr > Leu > He = Met > Trp = Phe > Val = Gin > Pro, Glu, Ser, Nph, Arg. Thus, most substitutions at subsite P l are detrimental relative to He. Interestingly, HNC generally tolerates aromatic amino acids better in this subsite than HFC. For subsite P 2, substitution of Ala by Phe, Trp, Leu, or Arg increases the rates of hydrolysis markedly for both enzymes. The substitution by Trp is most dramatic and causes increases in rate of 7.3- and 8.3-fold for HFC and HNC, respectively. This clearly demonstrates the importance of the interaction of the substrate side-chain in this subsite with these collagenases. Substitution by Glu lowers the rates for both enzymes, while Hyp abolishes measurable hydrolysis. 

Pretreatments of the polymeric feedstocks to enhance the hydrolysis of the major components such as cellulose include size reduction, thermal-chemical treatments, and specific enzyme pretreatments (52,57). Although many of the pretreatment processes increase the rate and extent of potymer hydrolysis, the cost for treatment is prohibitive based upon the revenues generated for energy production alone in anaerobic digestion (32). 

The stability of the ester surfactants against enzymatic hydrolysis by two different microbial Upases, Mucor miehei lipase (MML) and Candida antarc-tica lipase B (CALB) added separately to the surfactant solutions, was also investigated, see Fig. 5 [19]. It is obvious that hydrolysis of the unsubstituted surfactant is much faster with both CALB and MML than that of the substituted surfactants, i.e., increased steric hindrance near the ester bond leads to decreased hydrolysis rate. Since the specificity of the enzyme against its substrate is determined by the structure of the active site, it can be concluded, as expected, that the straight chain surfactant most easily fits into the active site of both enzymes. 

Activation of a-D-mannosidase from the limpet by Zn2+ and Cl-provides a particularly good example of the ways in which the kinetics of hydrolysis may be altered. Fig. 2 shows the effect of Zn2+, Cl-, or both, on the velocity of hydrolysis of substrate at varying concentration. Inspection of die curves reveals that Zn2+ increases the affinity of the enzyme for the substrate (competitive type of effect), whereas the main effect of Cl- is to increase the rate of hydrolysis (non-competitive effect). 

It has been shown that, in supercritical carbon dioxide, increases in water concentration result in increases in enzyme activity. The amount of added water needed for this increase varies and can depend on many factors, such as reaction type, enzyme utilized, and initial water content of the system. This is true until an optimal level is reached. For hydrolysis reactions, activity will either continue to increase or maintain its value. For esterification or transesterification reactions, once the optimal level of hydration has been reached, additional water will promote only side reactions such as hydrolysis. Dumont et al. (1992) suggests that additional water beyond the optimal level needed for enzyme hydration may also act as a barrier between the enzyme and the reaction medium and thereby reduce enzyme activity. Mensah et al. (1998) also observed that water above a concentration of 0.5 mmol/g enzyme led to lower catalytic activity and that the correlation between water content of the enzyme and reaction rate was independent of the substrate concentrations. 

The breakdown of fatty acids in (3-oxidation (see Topic K2) is controlled mainly by the concentration of free fatty acids in the blood, which is, in turn, controlled by the hydrolysis rate of triacylglycerols in adipose tissue by hormone-sensitive triacylglycerol lipase. This enzyme is regulated by phosphorylation and dephosphorylation (Fig. 5) in response to hormonally controlled levels of the intracellular second messenger cAMP (see Topic E5). The catabolic hormones glucagon, epinephrine and norepinephrine bind to receptor proteins on the cell surface and increase the levels of cAMP in adipose cells through activation of adenylate cyclase (see Topic E5). The cAMP allosterically activates... 

We have made several artificial enzymes that use cyclodextrin to bind a substrate and then react with it by acylating a cyclodextrin hydroxyl group. This builds on earlier work by Myron Bender, who first studied such acylations [83]. We added groups to the cyclodextrin that produced a flexible floor, capping the ring [84]. The result was to increase the relative rate of cyclodextrin acylation by m-t-butylphenyl acetate from 365 relative to its hydrolysis rate in the buffer to a Complex/ buffer of 3300. We changed the substrate to achieve better geometry for the intracomplex acylation reaction, and with a p-nitrophenyl ester of ferroceneacrylic acid 10 we achieved a relative rate for intracomplex acylation of ordinary [3-cyclodextrin vs. hydrolysis of over 50 000 and a Vmax comparable to that for hydrolysis of p-nitrophenyl acetate by chymotrypsin... 

The next section describes further mimics of ribonuclease enzymes. However, we mention here one additional study on the cleavage of ribonucleotides in which metal ions were used [123]. Cyclization of uridyluridine 39, and hydrolysis of the resulting cyclic nucleotides 40, was catalyzed by Eu3+ much more effectively than by Zn2+, and some added ligands increased the rates. 

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