The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of <i>N</i>‐acetyl‐α‐<scp>L</scp>‐amino acid methyl esters

Martínek, Karel; Klyosov, Anatole A.; Kazanskaya, N.F.; Березин, И.В.

doi:10.1002/kin.550060604

Cited by 11 publications

(1 citation statement)

References 23 publications

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“…Generally, these investigations were conducted either to demonstrate the existence of an acyl-enzyme intermediate (Bender et al, 1964;Berezin et al, 1971;Fersht et al, 1973) or to examine the utility of serine proteases as catalysts of amide bond formation in peptide synthesis (Jakubke et al, 1985;Martinek et al, 1974;Petkov & Stoineva, 1984;Riechmann & Kasche, 1985).…”

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Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides

Stein¹,

Strimpler²

1987

Biochemistry

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Acyl-enzymes of human leukocyte elastase (HLE) were generated in situ during the hydrolysis of peptide thiobenzyl esters and served as substrates for aminolysis by a variety of amino acid amides and short peptide nucleophiles. For amino acid amides, there is a positive correlation between nucleophilic reactivity toward N-methoxysuccinyl (MeOSuc)-Ala-Ala-Pro-Val-HLE and the hydrophobicity of the side chain. For peptides, nucleophilicity toward MeOSuc-Ala-Ala-Pro-Val-HLE decreases dramatically with increasing chain length. Combined, these results suggest that substrate specificity for the P1' residue may be more dependent on side chain hydrophobicity than on specific, structural features of the side chain and there may be no important binding interactions available past S1'. Kinetic parameters were also determined for the nucleophilic reactions of PheNH2 and TyrNH2 with MeOSuc-Pro-Val-HLE, MeOSuc-Ala-Pro-Val-HLE, MeOSuc-Ala-Ala-Pro-Val-HLE, and MeOSuc-Ala-Ala-Pro-Ala-HLE. Reactivity of these acyl-enzymes toward nucleophilic attack displays no dependence on peptide chain length but does increase significantly for the substrate with Ala at P1. This same correlation between reactivity and acyl-enzyme structure is also seen for nucleophilic attack by water.

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Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides

Stein¹,

Strimpler²

1987

Biochemistry

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A new approach to preparative enzymatic synthesis

Klibanov¹,

Samokhin²,

Martínek³

et al. 2000

Biotechnol. Bioeng.

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A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water‐immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water‐miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the systhesis of N‐acetyl‐L‐tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N‐acetyl‐L‐tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.

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A new approach to preparative enzymatic synthesis

Klibanov,

Samokhin,

Martinek

et al. 1977

Biotech & Bioengineering

184

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SummaryA new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-btryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-btryptophan and ethanol. In water (no organic phase) the yield of the ester is about O.Ol%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.

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The free energy–reaction coordinate profile for α‐chymotryptic hydrolysis of a series of N‐acetyl‐α‐L‐amino acid methyl esters

Cited by 11 publications

References 23 publications

Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides

Catalysis by human leukocyte elastase. Aminolysis of acyl-enzymes by amino acid amides and peptides

A new approach to preparative enzymatic synthesis

A new approach to preparative enzymatic synthesis

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