Binding of organic solvent molecules influences the P1??P2? stereo-and sequence-specificity of ?-chymotrypsin in kinetically controlled peptide synthesis

Kasche, Volker; Michaelis, Günther; Galunsky, Boris

doi:10.1007/bf01030454

Cited by 35 publications

(12 citation statements)

References 16 publications

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“…This specificity has now been studied quantitatively by determining the enantio-(or stereo-)selectivity of several enzymes -mainly for hydrolases (Chen et al, 1982Kasche et al, 1991). The selectivity is expressed as a ratio of rate constants, and was found to differ by orders of magnitude for different substrates using the same enzyme, or for different enzymes using the same substrate.…”

Section: Introductionmentioning

confidence: 99%

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S?1-subsites- on temperature and primary structure

et al. 1996

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The stereoselectivity of penicillin amidase (PA, EC 3.51 .ll) from E co/i and homologeous enzymes from other sources has been determined as a function of temperature and substrate for hydrolysis and kinetically controlled synthesis. The stereoselectivity of these reactions decreased almost by one order of magnitude from 5 to 45°C. It increased with the substrate (kcat/Km) and nucleophile (e/h) specificity, and was found to differ in the Sl-(R-specific) and S,l -(S-specific)-binding subsites of the active site. The Sl -stereoselectivity was determined mainly by differences in the activation energy, i.e. the turnover number. The stereoselectivity of PA from different sources differed by almost an order of magnitude for the same substrate.

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Section: Introductionmentioning

confidence: 99%

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S?1-subsites- on temperature and primary structure

et al. 1996

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“…Alternatively, acetonitrile may increase the binding of the nucleophile, possibly by causing conformational changes in the proteinase. Kasche et al 13 have shown that the binding of organic solvent molecules can influence the PI-P, stereo and sequence specificity of a-chymotrypsin catalyzed peptide synthesis, as mentioned earlier.…”

Section: Effect Of the Solvent Concentrationmentioning

confidence: 92%

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

Wilson¹,

Daniel

Peek

1994

Biotech & Bioengineering

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A proteinase isolated from Thermus RT41a was immobilized to controlled pore glass beads and was used in the free and immobilized forms for peptide synthesis. The observed maximum yield was the same in both cases. a number of dipeptides were produced from amino acid esters and amides. The best acyl components, from those tested, were found to be Ac-Phe-OEt and Bz-Ala-OMe. Tur-NH(2), Trp-NH(2), Leu-pNA, and Val-pNA were all reactive nucleophiles.The kinetically controlled synthesis of Bz-ala-Tyr-NH(2) was optimized by studying the effect of pH, temperature, solvent concentration, ionic strength, and nucleophile and acyl donor concentration, ionic strength, and nucleophile and acyl donor concentration on the maximum yield. The initial conditions used were 25 mM Bz-ala-OMe, 25 mM Tyr-NH(2), 70 degrees C, pH 8.0, and 10% v/v dimethylformamide (DMF). The optimum conditions were 90% v/v DMF using 80 mM bz-Ala-OMe and 615 mM Tyr-NH(2) at 40 degrees C and pH 10. These conditions increased the maximum conversion from 0.75% to 26% (of the original ester concentration). In a number of other cosolvents, the best peptide yields were observed with acetonitrile and ethyl acetate. In 90% acetonitrile similar yields were observed to those in 90% DMF under optimized conditions except that the acyl donor and nucleophile concentrations could be reduced to 25 mM and 100mM, respectively. The effect of the blocking group on the nucleophile was also investigated; -betaNA and -pNA as blocking groups improved the yields markedly. The blocking and leaving groups of the acyldonor had no effect on the dipeptide yield.

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“…While organic solvents are known to influence biocatalysts through direct solvent/enzyme molecular interactions (Affleck et al, 1992;Burke et al, 1993), solvent/binding site interactions (Kasche et al, 1991;Ryu and Dordick, 1992), and solvent/transition state interactions (Ke et al, 1996;Michels et al, 1997), the complexities of predicting such effects make correlations with solvent parameters such as those considered in Table I especially valuable. Moreover, solvent effects on the chemical potentials of soluble reactants can be addressed in a straightforward manner.…”

Section: Impact Of Solvent Environment On Reactant Activitiesmentioning

confidence: 99%

Solid‐phase peptide synthesis by ion‐paired α‐chymotrypsin in nonaqueous media

Altreuter

Dordick

Clark

2003

Biotech & Bioengineering

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Solid-phase synthesis of dipeptides in low-water media was achieved using AOT ion-paired alpha-chymotrypsin solubilized in organic solvents. Multiple solvents and systematic variation of water activity, a(w), were used to examine the rate of coupling between N-alpha-benzyloxycarbonyl-L-phenylalanine methyl ester (Z-Phe-OMe) and leucine as a function of the reaction medium for both solid-phase and solution-phase reactions. In solution, the observed maximum reaction rate in a given solvent generally correlated with measures of hydrophobicity such as the log of the 1-octanol/water partitioning coefficient (log P) and the Hildebrand solubility parameter. The maximum rate for solution-phase synthesis (13 mmol/h g-enzyme) was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.30. For the synthesis of dipeptides from solid-phase leucine residues, the highest synthetic rates (0.14-1.3 mmol/h g-enzyme) were confined to solvent environments that fell inside abruptly defined regions of solvent parameter space (e.g., log P > 2.3 and normalized electron acceptance index <0.13). The maximum rate for solid-phase synthesis was obtained in a 90/10 (v/v) isooctane/tetrahydrofuran solvent mixture at an a(w) of 0.14. In 90/10 and 70/30 (v/v) isooctane/tetrahydrofuran environments with a(w) set to 0.14, seven different N-protected dipeptides were synthesized on commercially available Tentagel support with yields of 74-98% in 24 h.

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Binding of organic solvent molecules influences the P1??P2? stereo-and sequence-specificity of ?-chymotrypsin in kinetically controlled peptide synthesis

Cited by 35 publications

References 16 publications

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S?1-subsites- on temperature and primary structure

The dependency of the stereoselectivity of penicillin amidases-enzymes with R-specific S1- and S-specific S?1-subsites- on temperature and primary structure

Peptide synthesis with a proteinase from the extremely thermophilic organism Thermus Rt41A

Solid‐phase peptide synthesis by ion‐paired α‐chymotrypsin in nonaqueous media

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