Categorization of enzyme deactivations using a series-type mechanism

Henley, James P.; Sadana, Ajit

doi:10.1016/0141-0229(85)90013-4

Cited by 220 publications

(117 citation statements)

References 36 publications

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“…The experimentally determined deactivation profiles were fitted using the above two-step model and also with a first-order deactivation model [23] ( Table 2 and Table 3). …”

Section: Biocatalyst Stabilitymentioning

confidence: 99%

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

2007

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A recombinant Escherichia coli strain was constructed which efficiently expressed the enantioselective nitrilase from Alcaligenes faecalis DSMZ 30030 as a hisitidine-tagged enzyme variant under the control of a rhamnose inducible promoter. The enzyme was purified from cell extracts and used for the preparation of cross-linked enzyme aggregates (CLEAs). The conditions for the preparation of the CLEAs were optimized using various organic solvents and cross-linking agents and a procedure was developed which combined a precipitation with 85 % (v/v) isopropyl alcohol and a cross-linking with 30 mM glutaraldehyde. Thus, about 80 % of the initial nitrilase activity could be incorporated into the CLEAs. The hydrolysis of racemic mandelonitrile to (R)-mandelic acid was compared between the soluble nitrilase preparations and their CLEAs (nitCLEAs). The nitrilase activity in the CLEAs was at 30 8C and 608C about 5 times more stable than in the soluble preparations. The CLEAs could be reused 5 times with only about 10 % reduction in activity. The enantioselectivity of the nitrilase for the formation of (R)-mandelic acid from racemic mandelonitrile decreased for both preparations with increasing temperatures (10 8C to 508C), but this effect was significantly less pronounced for the CLEAs. A detailed analysis of solvent effects on nitrilase enantioselectivity allowed thermodynamic insights into contributions from free energy component (activation enthalpy and entropy) to chiral preference of nitrilase in such non conventional media.

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“…The experimentally determined deactivation profiles were fitted using the above two-step model and also with a first-order deactivation model [23] ( Table 2 and Table 3). …”

Section: Biocatalyst Stabilitymentioning

confidence: 99%

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

2007

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“…This occurs when the inhibitor binds at a site away from the substrate binding site, causing a reduction in the catalytic rate. It is quite rarely found as a special case of mixed inhibition (Henley & Sadana, 1985;Hill et al, 1977;Koshland, 1962). The fractional inhibition is identical at all substrate concentrations and cannot be overcome by increasing substrate concentration due to the reduction in Vmax.…”

Section: E+s Es Essmentioning

confidence: 99%

Interactions of Fungicides and Pesticides with Specific Enzymes

Ekinci¹,

Şentürk²

2010

Fungicides

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“…At regular intervals of time, 5-l aliquots were extracted from the incubation mixture and the residual activity was determined at 40°C using 2Ј-deoxyadenosine synthesis from 2Ј-deoxyuridine and adenine under the standard assay conditions. The experimental deactivation data were modeled by equation 1 or equation 2 using a nonlinear regression procedure based on the Lvenberg-Marquardt method of iterative convergence (24) included in the TableCurve 2D v5.01 program:…”

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confidence: 99%

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

Fernández‐Lucas

Acebal

Sinisterra³

et al. 2010

Appl Environ Microbiol

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A novel type II nucleoside 2-deoxyribosyltransferase from Lactobacillus reuteri (LrNDT) has been cloned and overexpressed in Escherichia coli. The recombinant LrNDT has been structural and functionally characterized. Sedimentation equilibrium analysis revealed a homohexameric molecule of 114 kDa. Circular dichroism studies have showed a secondary structure containing 55% ␣-helix, 10% ␤-strand, 16% ␤-sheet, and 19% random coil. LrNDT was thermostable with a melting temperature (T m ) of 64°C determined by fluorescence, circular dichroism, and differential scanning calorimetric studies. The enzyme showed high activity in a broad pH range (4.6 to 7.9) and was also very stable between pH 4 and 7.9. The optimal temperature for activity was 40°C. The recombinant LrNDT was able to synthesize natural and nonnatural nucleoside analogues, improving activities described in the literature, and remarkably, exhibited unexpected new arabinosyltransferase activity, which had not been described so far in this kind of enzyme. Furthermore, synthesis of new arabinonucleosides and 2-fluorodeoxyribonucleosides was carried out.Nucleoside 2Ј-deoxyribosyltransferases (NDTs) (EC 2.4.2.6) catalyze the exchange between the purine or pyrimidine base of 2Ј-deoxyribonucleosides and free pyrimidine or purine bases (10, 25). These enzymes are specific for 2Ј-deoxyribonucleosides, regioselective (N-1 glycosylation in pyrimidine and N-9 in purine), and stereoselective (␤-anomers are exclusively formed) (26) (Fig. 1).Deoxyribosyltransferases are classified into two classes depending on their substrate specificity: type I (NDT I), specific for purines (Pur 7 Pur), and type II (NDT II), which catalyzes the transfer between purines and/or pyrimidines (Pur 7 Pur, Pur 7 Pyr, Pyr 7 Pyr) (10, 25). These enzymes were initially described for lactobacilli (27,28), and they are involved in the nucleoside salvage pathway for DNA synthesis (23), although this remains unclear in Lactococcus lactis subsp. lactis (36). NDTs have been also found in some species of Streptococcus (11), in parasitic unicellular eukaryotic organisms such as Crithidia luciliae (49, 50), in Trypanosoma brucei (6), and in Borrelia burgdorferi (33). NDTs from Lactobacillus helveticus and Lactobacillus leichmannii have been well studied (2,25,26,28,29), and their kinetic mechanisms as well as their catalytic and substrate binding sites have been characterized. The transferase reaction proceeds via a ping-pong bi-bi mechanism by formation of a covalent deoxyribosyl enzyme intermediate (3,15,16). Likewise, a glutamyl residue (Glu98) has been proven essential for activity (40,41,46). Enzymatic natural and nonnatural nucleoside synthesis in a one-pot reaction by NDTs provides an interesting alternative to traditional multistep chemical methods (13,34). Indeed, chemical glycosylation includes several protection-deprotection steps and the use of chemical reagents and organic solvents that are expensive and environmentally harmful. Whereas previously described NDTs accept different nucleosides fr...

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Categorization of enzyme deactivations using a series-type mechanism

Cited by 220 publications

References 36 publications

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

Cross‐Linked Amorphous Nitrilase Aggregates for Enantioselective Nitrile Hydrolysis

Interactions of Fungicides and Pesticides with Specific Enzymes

Lactobacillus reuteri 2′-Deoxyribosyltransferase, a Novel Biocatalyst for Tailoring of Nucleosides

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