Michaelis-Menten kinetics, ester hydrolysis

Lipase hydrolysis of emulsified esters in water does not obey simple Michaelis-Menten kinetics. The hydrolysis of water-insoluble substrates involves the adsorption of the lipase to the substrate-water interface [5]. This adsorption is in many cases connected... 

Results have generally been disappointing. It can be difficult to remove the TSA from the polymer, but a more fundamental problem concerns the efficiency of the catalysis observed. The most efficient systems catalyze the hydrolysis of carboxylate and reactive phosphate esters with Michaelis-Menten kinetics and accelerations (koAJKM)/kunoJ approaching 103,1661 but the prospects for useful catalysis of more complex reactions look unpromising. Apart from the usual difficulties the active sites produced are relatively inflexible, and the balance between substrate binding and product inhibition is particularly acute. 

In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

We conclude that the neutral substrate enters 1 to form a host-guest complex, leading to the observed substrate saturation. The encapsulated substrate then undergoes encapsulation-driven protonation, presumably by deprotonation of water, followed by acid-catalyzed hydrolysis inside 1, during which two equivalents of the corresponding alcohol are released. Finally, the protonated formate ester is ejected from 1 and further hydrolyzed by base in solution. The reaction mechanism (Scheme 7.7) shows direct parallels to enzymes that obey Michaelis-Menten kinetics due to the initial pre-equilibrium followed by a first-order rate-limiting step. 

The MIP-mediated hydrolysis of ester 70 in HEPES buffer (0.15 M, pH 7.3) and MeCN at 20 °C showed Michaelis-Menten kinetics and a 325-fold rate enhancement compared to the reaction performed in solution. By comparison, the hydrolysis of ent-70 showed a 234-fold enhancement under the same conditions, resulting in a pronounced enantioselectivity. Interestingly, the apparently slight difference between 70 and 71 resulted in a significant decrease in catalyst activity, as the rate enhancement compared to the reaction performed in solution was only 103-fold. 

A transesterication reaction occurs that results in cleavage of the substrate and ligation of the 3 -portion of the substrate (Tsang and Joyce 1994). Just like in the case of enzyme- or catalytic antibody-catalyzed reactions, the rate depends upon substrate binding affinity and the intrinsic catalytic rate parameters. For example, in ester hydrolysis there is a hyperbolic dependence on the concentration of the ribozyme at low concentration of catalyst the rate of hydrolysis is first order, while at high concentration of catalyst the reaction rate is indepen-dent of ribozyme concentration (Piccirilli et al. 1992). This type of saturation or Michaelis-Menten kinetic behavior is typical of ribozymes and is completely analogous to the enzyme-substrate complex observed for enzymes and catalytic antibodies. 

Catalysis. Many reactions are catalyzed, i.e., increased in rate, by a compound in solution (homogeneous catalysis) or a group at the surface of a particle (heterogeneous catalysis), where the catalyst is not consumed itself. Examples are various hydrolyzing reactions, like the ester hydrolysis mentioned above, that are catalyzed by H+ as well as OH ions. In such a case the reaction rate greatly depends on pH, though the ions themselves do not appear as reactants in the overall reaction scheme. Ubiquitous in natural foods are enzyme-catalyzed reactions. The simplest case leads to Michaelis-Menten kinetics, but several complications may arise. 

Macrolides have virtually no ring strain, and hence, show similar reactivities with acyclic fatty acid alkyl esters in the alkaline hydrolysis and lower anionic ring-opening polymerizabihty than e-CL. However, polymerization of the macrolides using lipase PF catalyst proceeded much faster than that of -CL. This speciflc polymerizabihty by hpase catalyst was quantitatively evaluated by Michaelis-Menten kinetics (160,168,170-172). For imsubstituted lactones in the... 

Under mildly alkaline conditions and in the presence of excess cyclo-heptaamylose the rate of degradation of penicillin is increased 2(U90-fold compared with the rate of alkaline hydrolysis (Tutt and Schwartz, 1971). Michaelis-Menten kinetics are observed which are indicative of complex formation. The apparent binding constant of 6-substituted penicillins varies little with the length of the alkyl side chain although it is increased about 10-fold for diphenylmethyl penicillin. The reaction is catalytic and hydrolysis proceeds by the formation of a penicilloyl- 3-cyclodextrin covalent intermediate, i.e. ester formation, by nucleophilic attack of a carbohydrate hydroxyl on the P-lactam. 

The most widely used strategy involves the synthesis of the network around a structural analogue of the transition state of the reaction. The imprinted sites then correspond to the conformation of the substrates in the transition state. For ester hydrolysis this state can, for instance, be simulated by a phosphonate derivative as template [156,167]. An imprinted network with an esterase-type catalytic activity can then be obtained. For the MIP represented in Fig. 19(1), the reaction rate is increased 100-fold with respect to the reaction without catalyst and kinetics of the Michaelis-Menten type, as well as inhibition by an analogue of the transition state are observed [156]. 

Schematically shown in Fig. 5 is the preparation of an enzyme mimic for the hydrolysis of ester 6 by molecular imprinting. Phosphonate 5 is an analog of the transition state for the alkaline hydrolysis of Ester 4. It was used as a template for polymerization with two equivalents of the binding-site monomer iVJV -diethyl-4-vinyl-benzamidine. Amidinium groups were chosen, because they can interact electrostatically with the side carboxyl-ate group as well as with the anionic transition state of the alkaline hydrolysis, thus achieving substrate recognition and transition-state stabilization. Polymerization of the preassembled binding-site monomer with the template (Fig. 5A) followed by template removal (Fig. 5B) leaves a cavity that acts as transition-state receptor for the ester substrate (Fig. 5C). The imprinted polymer accelerates the hydrolysis of 6 more than 100-fold compared to the reaction at the same pH in buffer solution without the polymer. The reaction kinetics is of the Michaelis-Menten type. A polymer obtained with amidinium benzoate as a control, with a statistical distribution of amidinium groups, is ca. one order of magnitude less active in the hydrolysis of 6.

In ester synthesis and exchange reactions, as well as in hydrolysis re tions induced by PEG-lipase in hydrophobic media, the existence of a trace amount of water in the reaction system was most important in terms of the reactions proceeding. Matsushima et al. [67] carried out a kinetics study of PEG-lipase in transparent benzene solution to estimate the value of water, one of the substrates of lipase in the ester hydrolytic reaction. Indoxyl acetate was hydrolyzed by PEG-lipase to form acetic acid and 3-hydroxyindole, which was photometrically determined. A double-reciprocal plot of the velocity of the indoxyl acetate hydrolysis against water concentration at a given concentration of indoxyl acetate indicated that the hydrolysis took place as a double-displacement reaction (ping-pong reaction). The apparent Michaelis-Menten constant of water and the maximum velocity were calculated to be = 7 X 10 M and Vmax = 4700 xmol/min/mg of protein, respectively. 

We will consider first an enzyme-catalyzed reaction where there is a single substrate. An example is the hydrolysis of an ester. The dependence on substrate concentration in such cases is frequently as shown in Figure 10.1. The rate varies linearly with the substrate concentration at low concentrations (first-order kinetics), and becomes independent of substrate concentration at high concentrations (zero-order kinetics). This type of behavior was first explained in 1913 by the German-American chemist Leonor Michaelis (1875-1949) and-his Canadian assistant Mary L. Menten in terms of the mechanism... 

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