Regular ArticleKinetic Study of the α-Chymotrypsin-Catalyzed Hydrolysis and Synthesis of a Peptide Bond in a Monophasic Aqueous/Organic Reaction Medium
Abstract
We have studied the hydrolysis and synthesis reactions of the peptide bond involved in N-Cbz-L-tryptophanylglycineamide as catalyzed by α-chymotrypsin in various mixtures of water and 1,4-butanediol. Using a constant nonsaturating concentration of substrate, the initial reaction rates decreased exponentially with decreasing water content in the solvent mixture. When the water content was decreased from 100 to 20% (v/v), the maximum rate of reaction did not vary by more than a factor of 2 to 4, whereas the Michaelis constant increased exponentially. This exponential variation of the Michaelis constant was due to changes in the partitioning of the substrate between the active site of the enzyme and the solvent. In these processes, the actual rate constants did not vary when the relative contents of water and 1,4-butanediol were varied.
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Kinetics of oxidation of hydrogen peroxide at hemin-modified electrodes in nonaqueous solvents
2009, BioelectrochemistryHemin adsorbed on graphite electrodes and used to catalyse the reduction of hydrogen peroxide in an aqueous buffer and in a range of nonaqueous solvents has been described. The immobilised hemin is stable in the solvents examined. The rate limiting step involves the reaction between hemin and hydrogen peroxide. Kinetic analysis of the response in nonaqueous solvents showed that Imax / Kmapp increased linearly with the solvent hydrophobicity (log P) in all solvents, a trend that is explained by preferable partitioning of hydrogen peroxide into the polar hemin layer.
Biological processes in organised media
2003, Comptes Rendus - BiologiesEmbedding a simple Michaelis–Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach. To cite this article: M. Thellier et al., C. R. Biologies 326 (2003).
La fixation d'enzymes michaéliennes dans une lame de gel peut suffire à les rendre aptes à catalyser des processus, non seulement scalaires, mais également vectoriels, y compris le transport actif d'un substrat entre deux compartiments, et à les faire se comporter comme le feraient un double mécanisme enzymatique ou de transport ou un processus allostérique. Dans un environnement partiellement hydrophobe, les paramètres cinétiques apparents n'ont en général rien à voir avec les véritables paramètres caractéristiques du comportement de la même protéine en solution aqueuse. Réciproquement, lorsque l'on observe (en coordonnées inverses) que des systèmes enzymatiques ou de transport in vivo s'ajustent mieux à deux approximations linéaires ou à une courbe sigmoı̈de qu'à une seule droite, ceci ne prouve pas qu'interviennent deux systèmes enzymatiques ou de transport différents ou un processus allostérique. Avec les systèmes de transport étudiés ici, il est apparu qu'une asymétrie fonctionnelle pouvait suffire à induire un transport à contre-gradient en l'absence de toute asymétrie structurale ; aussi, bien que tous les systèmes de transport actif isolés jusqu'ici correspondent à des protéines à structure asymétrique par rapport à la membrane où elles sont insérées, on ne peut pas exclure que des systèmes de transport à structure symétrique existent ou aient existé au cours de l'évolution. Avec des systèmes réels, tel que celui qui maintient le potentiel électrique de la peau de grenouille, augmenter la complexité du comportement du système par l'induction d'oscillations électriques par addition de lithium a permis d'atteindre des données sur ce système qu'il n'aurait pas été possible d'obtenir par l'approche réductionniste traditionnelle. Pour citer cet article : M. Thellier et al., C. R. Biologies 326 (2003).
Enzymes which are stable in the presence of organic solvents
2001, Journal of Bioscience and BioengineeringThere are numerous advantages of employing enzymes as catalysts in organic solvents or aqueous solutions containing organic solvents instead of water. A few natural enzymes which are stable in the presence of organic solvents have been discovered. However, almost all natural enzymes are easily denatured and inactivated in the presence of organic solvents. Therefore, several physical and chemical methods, such as immobilization, modification, and entrapment, for stabilizing enzymes in the presence of organic solvents were developed. Protein engineering using site directed mutagenesis and directed evolution are useful for clarifying why organic solvent-stable enzymes are stable in the presence of organic solvents and for developing organic solvent-stable mutant enzymes.
Peptide synthesis catalyzed by organic solvent-stable protease from Pseudomonas aeruginosa PST-01 in monophasic aqueous-organic solvent systems
1999, Journal of Bioscience and BioengineeringThe equilibrium yields of the peptide Cbz-Arg-Leu-NH2 synthesized from Cbz-Arg and Leu-NH2 using the PST-01 protease in the presence of organic solvents were investigated under various conditions. The equilibrium yields depended little on the concentration of the carboxyl component, but significantly on the concentration of the nucleophile. The optimum temperature and pH for a high equilibrium yield were 30°C and greater than 5.0, respectively. Under optimum conditions the equilibrium yields were 71.6% and 87.7% in the presence of 50% (v/v) DMF and 60% (v/v) DMSO, respectively. Furthermore, the PST-01 protease also catalyzed the syntheses of the dipeptides Cbz-Lys-Leu-NH2, Cbz-Ala-Leu-NH2, Cbz-Ala-Phe-NH2, Cbz-Arg-Leu-NH2, and Cbz-Lys-Phe-NH2 with equilibrium yields of more than 60% in the presence of 50% (v/v) DMF and 50 mM sodium phosphate buffer (pH 7.0).
Peptide synthesis using proteases dissolved in organic solvents
1997, Enzyme and Microbial TechnologyOrganic solvent-soluble α-chymotrypsin (CT) and subtilisin Carlsberg (SC) are effective catalysts for peptide synthesis in homogeneous organic solutions. The soluble enzymes have values of for the reaction of N-Bz-L-Tyr-OEt with L-Leu-NH2 to yield the dipeptide N-Bz-L-Tyr-L-Leu-NH2 that are over 3 orders of magnitude higher than their suspended counterparts in isooctane (containing 30% (v/v) tetrahydrofuran (THF) to aid in substrate solubility). Both enzymes are substantially more active in hydrophobic organic solvents than hydrophilic solvents. Adding small concentrations of water (<0.2% and 1% (v/v) in isooctane-THF and ethyl acetate, respectively) results in up to a 150-fold activation of α-chymotrypsin-catalyzed peptide synthesis. Importantly, added water does not promote hydrolysis in either isooctane-THF or ethyl acetate; thus, α-chymotrypsin is highly selective toward peptide synthesis in the nearly anhydrous organic solutions. Unlike CT, the activation of subtilisin Carlsberg upon partial hydration of isooctane-THF or ethyl acetate was not significant and actually resulted in substantial hydrolysis. Using α-chymotrypsin, a variety of tripeptides were produced from dipeptide amino acid esters. Reactivity of D-amino acid amides as acyl acceptors and partially unblocked amino acid acyl donors further expands the generality of the use of organic solvent-soluble enzymes as peptide synthesis catalysts.
Kinetics and Mechanism of a Reaction Catalyzed by PST-01 Protease from Pseudomonas aeruginosa PST-01
2004, Biotechnology and Bioengineering