Kinetic hydrolases

Some G proteins are slow GTP hydrolases with turnover numbers around two per minute, others such as Ras are only marginally catalytic. Kinetic experiments in solution have shown that in both cases the most likely mechanism... 

Enzyme Kinetics and Stability Kinetic studies, carried out mostly with hydrolases, have shovm that enzymes in organic solvents follow conventional models [12a, 22]. 

Several reports regarding the directed evolution of enantioselective epoxide hydrolases (EHs) have appeared [23,57-59]. These enzymes constitute important catalysts in synthetic organic chemistry [4,60]. The first two reported studies concern the Aspergillus niger epoxide hydrolase (ANEH) [57,58]. Initial attempts were made to enhance the enantioselectivity of the AN E H -catalyzed hydrolytic kinetic resolution of glycidyl phenyl ether (rac-19). The WT leads to an Evalue of only 4.6 in favor of (S)-20 (see Scheme 2.4) [58]. 

Figure 2.15 Iterative CASTing in the evolution of enantioseiective epoxide hydrolases as catalysts in the hydrolytic kinetic resolution ofrac-19[23].

In an asymmetric synthesis, the enantiomeric composition of the product remains constant as the reaction proceeds. In practice, ho vever, many enzymatic desymmetrizations undergo a subsequent kinetic resolution as illustrated in Figure 6.5. For instance, hydrolysis of a prochiral diacetate first gives the chiral monoalcohol monoester, but this product is also a substrate for the hydrolase, resulting in the production of... 

Comparison of whole cell biocatalytic reaction kinetics for recombinant Escherichia coli with periplasmic-secreting or cytoplasmic-expressing organophosphorus hydrolase... 

Pure human CESs (hCEl and hCE2), a rabbit CES (rCE), and two rat CESs (Hydrolases A and B) were used to study the hydrolytic metabolism of the following pyrethroids lR-trans-resmethrin (bioresmethrin), 1RS-1runs-permethrin, and 1RS-r/.v-pennethrin [28], hCEl and hCE2 hydrolyzed /ram-pemiethrin 8- and 28-fold more efficiently than m-permethrin (when kcat/Km values were compared), respectively. In contrast, hydrolysis of bioresmethrin was catalyzed efficiently by hCEl, but not by hCE2. The kinetic parameters for the pure rat and rabbit CESs were qualitatively similar to the human CESs when hydrolysis rates of the investigated pyrethroids were evaluated. Further, a comparison of pyrethroid hydrolysis by hepatic microsomes from rats, mice, and humans indicated that the rates for each compound were similar between species. 

Many studies have been performed in laboratory animals to better characterize the distribution, nature, regulation, kinetic properties, and substrate specificity of aspirin hydrolases, as they are sometimes designated (e.g., [41] [84-86]). 

A number of synthetic fatty acid thioesters, notably derivatives of branched fatty acids or thioester-containing phospholipids, have been used as kinetic and catalytic probes of various hydrolases [148] [149]. However, such compounds are of limited interest in our context. 

The enzymatic hydration of lactones is also documented, a variety of hydrolases having demonstrated activity. Very detailed kinetic studies have, for example, been published on the hydrolysis of oxazolones (7.78, R = H or Me, R = Me or aryl, R" = Me or Ph) catalyzed by a-chymotrypsin [163], These compounds are interesting from a chemical point of view, being enolic lac-... 

G. Bellucci, C. Chiappe, G. Ingrosso, Kinetics and Stereochemistry of the Microsomal Epoxide Hydrolase-Catalyzed Hydrolysis of cw-Stilbene Oxides , Chirality 1994, 6, 577 - 582. 

Most kinetic resolutions of pharmaceutical intermediates that have been reported involve the use of hydrolases, particularly lipases and proteases. This is because many hydrolases are commercially available (in bulk and kit form), do not require cofactors and are active in many organic solvents (see Section 1.4). Processes can therefore, often be developed rapidly, using high substrate concentrations and without specialist knowledge. 

Finally, one should recognize that determinations of the critical concentration depend wholly on the validity of the equilibrium or steady-state assumptions. If a stable end point for prdtomer-polymer coexistence is not attained, then kinetic factors affect the observed behavior. With the well recognized tendency of tubulin to lose its ability to engage in assembly reactions upon storage even at low temperature, and with the presence of various nucleotide hydrolases and transphosphorylases in microtubule protein, such kinetic effects are a serious problem. 

As discussed above, proteases are peptide bond hydrolases and act as catalysts in this reaction. Consequently, as catalysts they also have the potential to catalyze the reverse reaction, the formation of a peptide bond. Peptide synthesis with proteases can occur via one of two routes either in an equilibrium controlled or a kinetically controlled manner 60). In the kinetically controlled process, the enzyme acts as a transferase. The protease catalyzes the transfer of an acyl group to a nucleophile. This requires an activated substrate preferably in the form of an ester and a protected P carboxyl group. This process occurs through an acyl covalent intermediate. Hence, for kineticmly controlled reactions the eii me must go through an acyl intermediate in its mechanism and thus only serine and cysteine proteases are of use. In equilibrium controlled synthesis, the enzyme serves omy to expedite the rate at which the equilibrium is reached, however, the position of the equilibrium is unaffected by the protease. 

Dynamic Kinetic Resolution (DKR) of Azlactones Thioureas Can Act as Oxyanion Holes Comparable to Serine Hydrolases... 

This broad class of hydrolases constitutes a special category of enzymes which bind to and conduct their catalytic functions at the interface between the aqueous solution and the surface of membranes, vesicles, or emulsions. In order to explain the kinetics of lipolysis, one must determine the rates and affinities that govern enzyme adsorption to the interface of insoluble lipid structures -. One must also account for the special properties of the lipid surface as well as for the ability of enzymes to scooC along the lipid surface. See specific enzyme Micelle Interfacial Catalysis... 

Alice et al studied the turnover kinetics of Listeria OTonocytogenex-secreted p60 protein (a murein hydrolase) by host cell cytosolic proteasomes. J774 cells, seeded in flasks and incubated overnight in culture medium, were infected with log-phase cultures of E. monocytogenes for 30 min, washed, and incubated in culture medium for 3 h, with gentamicin (50 tg/ml) added after the first 30 min to inhibit extracellular bacterial growth. Cells then were washed and placed in methionine-free medium with spectinomycin, gentamicin, the eukaryotic protein synthesis inhibitors [cycloheximide (50 tg/mL) and anisomycin (30 tg/ml),] and 25 dVI calpain inhibitor I. After 30 min, [ S]methionine was added, and the cells were pulse-labeled for periods of 20 to 60 min. Cells... 

AMINO ACID RACEMASE Amino adds, peptides proteins, BIOCHEMICAL NOMENCLATURE AMINO ACID TURNOVER KINETICS AMINOACYLASE AMINOACYL-tRNA HYDROLASE Aminoacyl-tRNA synthetase,... 

HYDROGEN TUNNELING KINETIC ISOTOPE EEEECTS HYDROGEN TUNNELING HYDROLASES... 

There are basically two approaches to the synthesis of enantiomerically pure alcohols (i) kinetic resolution of the racemic alcohol using a hydrolase (lipase, esterase or protease) or (ii) reduction mediated by a ketoreductase (KRED). Both of these processes can be performed as a cascade process. The first approach can be performed as a dynamic kinetic resolution (DKR) by conducting an enzymatic transesterification in the presence of a redox metal [e.g. a Ru(ll) complex] to catalyze in situ racemization of the unreacted alcohol isomer [11] (Scheme 6.1). We shall not discuss this type of process in any detail here since it forms the subject of Chapter 1. 

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