| 1. |
- Gololobov, Mikhail Yu, et al.
(författare)
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Nucleophile specificity in α-chymotrypsin- and subtilisin-(Bacillus subtilis strain 72) catalyzed reactions
- 1992
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Ingår i: BBA - Protein Structure and Molecular Enzymology. - 0167-4838. ; 1160:2, s. 188-192
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Tidskriftsartikel (refereegranskat)abstract
- Nucleophilic properties of amino-acids were studied systematically in acyl-transfer reactions catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) using Mal-l-Ala-l-Ala-l-PheOMe as the acyl-group donor. In α-chymotrypsin-catalyzed reactions, the nucleophile reactivities increase in the following order: d-AlaNH2 < GlyNH2 < l-AlaNH2 < l-ScrNH2 < l-ThrNH2 < l-HisNH2 < l-ValNH2 < l-LcuNH2 < l-TrpNH2 < l-MetNH2 < l-NvaNH2 < l-PheNH2 < l-IleNH2 < l-TyrNH2 < l-ArgNH2. In reactions catalyzed by subtilisin 72, the reactivities increase as follows: l-LeuNH2 < l- IleNH2 < l-ThrNH2 < l-ArgNH2 < l-TrpNH2 < l-NvaNH2 < l-ValNH2 < l-MctNH2 < l-AlaNH2 < l-ScrNH2 < d-AlaNH2 < GlyNH2. In α-chymotrypsin-catalyzed reactions, hydrophobic interactions are entirely responsible for the differences between the reactivity of the nucleophiles for amides of all the amino-acids tested with the exception of d-AlaNH2, l-ArgNH2 and l-TyrNH2. In the reactions catalyzed by subtilisin 72, Amino-acid side-chain characteristics and the nucleophile reactivities are not related. The data obtained show the low selectivity of the S1′ subsite of subtilisin 72 and high specify of this subsite in α-chymotrypsin.
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| 2. |
- Gololobov, Mikhail Yu, et al.
(författare)
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Organic solvent changes the chymotrypsin specificity with respect to nucleophiles
- 1992
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Ingår i: FEBS Letters. - 0014-5793. ; 307:3, s. 309-312
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Tidskriftsartikel (refereegranskat)abstract
- In α-chymotrypsin-catalyzed acyl-transfer reactions in water the specificity of the enzyme (the nucleophile reactivity of amino acid amides) is correlated with the substrate hydrophobicity and increases as the hydrophobicity of the side chain of the amino acid amides is increased. In a low water system (4% H2O) bulky amino acid amides are less efficient nucleophiles. The specificity of α-chymotrypsin towards the amino acid amides in acyl transfer reactions in this case does not depend on the hydrophobicity of the amino acid side chains but correlates with their size. Therefore, different factors can be responsible for the specificity of enzymes in water and in a mainly organic medium.
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| 3. |
- Gololobov, Mikhail Yu, et al.
(författare)
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Side reactions in enzymatic peptide synthesis in organic media : Effects of enzyme, solvent, and substrate concentrations
- 1994
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 16:6, s. 522-528
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Tidskriftsartikel (refereegranskat)abstract
- The progress of enzymatic peptide synthesis catalyzed by α-chymotrypsin and subtilisin from Bacillus subtilis strain 72 (subtilisin 72) in low-water systems was studied. The initial reaction mixture consisted of the solvent, the acyl-group donor (MalAlaAlaPheOMe or ZAlaAlaPheOMe, Mal, maleyl, Z, benzyloxycarbonyl), the nucleophile XaaNH2 (Xaa = Phe, Leu or Ala), and the enzyme adsorbed on porous silica material. All amino acid residues were of the l-configuration. The solvent consisted of acetonitrile, dimethylformamide (DMF), and 4% (v/v) of water. The DMF/acetonitrile ratio was varied between 0 and 1/1. At high concentration of the acyl-group donor and approximately equimolar ratio of the nucleophile and the acyl-group donor, quantitative formation of MalAlaAlaPheXaaNH2 or ZAlaAlaPheXaaNH2 occurred. As a result, a method for the synthesis of polypeptide amides was developed. At low concentration of the acyl-group donor and excess of the nucleophile, the condensation by-products with two and three nucleophile molecules were found in the reaction mixtures. The data obtained provided evidence that organic solvents affected the S'1-specificity of α-chymotrypsin and the Sl-specificity of subtilisin 72, while the Sl-specificity of α-chymotrypsin and the S'l-specificity of subtilisin 72 were not affected. When the DMF content was increased, the rate of the α-chymotrypsin-catalyzed reactions decreased. In contrast to this, an increase in DMF content accelerated the subtilisin 72-catalyzed reactions. Hydrolysis of the acyl-group donor did not occur in the α-chymotrypsin-catalyzed reactions. Significant (up to 50%) formation of MalAlaAlaPheOH was observed a the early stage of the subtilisin 72-catalyzed reactions. Later MalAlaAlaPheOH underwent synthesis.
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| 4. |
- GOLOLOBOV, Mikhail Y., et al.
(författare)
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The second nucleophile molecule binds to the acyl‐enzyme–nucleophile complex in α‐chymotrypsin catalysis : Kinetic evidence for the interaction
- 1993
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 217:3, s. 955-963
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Tidskriftsartikel (refereegranskat)abstract
- α‐Chymotrypsin‐catalyzed acyl tranfer was studied using three acyl‐group donors (Mal‐l‐Ala‐l‐Ala‐l‐PheOMe, Bz‐l‐TyrOEt and Ac‐l‐TrpOEt; Mal, maleyl; Bz, benzoyl; OMe, methyl ester; OEt, ethyl ester) and a series of amino‐acid amides. Most of the reactions studied can be described by the simplest kinetic model without the nucleophile binding to the acyl‐enzyme. The α‐chymotrypsin‐catalyzed transfer of the Mal‐l‐Ala‐l‐Ala‐l‐Phe group to the amides of L‐Phe and L‐Tyr showed a linear dependence of the partition constant, p, on the nucleophile concentration which can be interpreted by the hydrolysis of the acyl‐enzyme–nucleophile complex. The α‐chymotrypsin‐catalyzed transfer of the Bz‐l‐Tyr and Ac‐l‐Trp groups to several amino‐acid amides showed unusual behavior which can be interpreted by the kinetic model involving formation of a complex of the acyl‐enzyme with two nucleophile molecules. These observations can explain the conflicting conclusions concerning the kinetics of α‐chymotrypsin‐catalyzed acyl transfer evident in previous studies.
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