Kinetic isotope effect investigation of enzyme mechanisms in organic solvents

Adams, K. A. H.; Chung, Soo Il; Klibanov, Alexander M.

doi:10.1021/ja00181a068

Cited by 29 publications

(15 citation statements)

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“…That the structures of the crystalline acyl-enzyme intermediate formed in acetonitrile and in water are the same verifies earlier kinetic evidence (21)(22)(23)(24) that the enzymatic mechanisms in organic solvents and in water are similar. The observed distinct binding of solvent molecules in the enzyme active site between the acyl-enzyme intermediate and the free enzyme structures in different solvents may be responsible for at least some of the heretofore unexplained disparity in the activity of enzymes in organic solvents.…”

Section: Resultssupporting

confidence: 84%

“…Because subtilisin exhibits markedly different catalytic activity and other properties in different solvents (7)(8)(9)(10)(11)(12)(13)(14)(21)(22)(23)(24), the structure of the active site in these solvents is a critical issue. We find that not only is the overall structure of the crystalline acyl-enzyme the same whether formed in acetonitrile or in water, but it can be seen in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…4D). This gives rise to the question; can the dissimilar solvation of the active site in different solvents be a contributor to the distinct subtilisin activities in them (21)(22)(23)(24)? Serine proteases such as subtilisin act by means of a ping-pong mechanism (40), where the ''pong'' is the deacylation step.…”

Section: Resultsmentioning

confidence: 99%

“…In organic solvents, however, the formation of such an intermediate in the catalysis by subtilisin is supported by kinetic but not direct structural data (21)(22)(23)(24).…”

mentioning

confidence: 99%

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Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Schmitke

Stern

Klibanov

1998

Proc. Natl. Acad. Sci. U.S.A.

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The x-ray crystal structures of trans-cinnamoyl-subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl-subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl-and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.Enzymes in organic solvents, instead of their natural aqueous milieu, remain synthetically useful catalysts (1-6). In addition, they display remarkable properties, e.g., solvent-dependent selectivity (7,8). Such solvent dependences of prochiral selectivity (9) and enantioselectivity (10) of crystalline enzymes in nonaqueous media have been nearly quantitatively predicted using structure-based molecular modeling and thermodynamic calculations.Despite recent advances (11)(12)(13)(14), the question of why enzymatic activity is often much reduced in organic solvents compared with water is far from resolved. Such an understanding, like that concerning the selectivity, requires both structural and mechanistic information. Recently, structures of several enzymes in neat organic solvents, namely those of subtilisin Carlsberg in acetonitrile (15, 16) and dioxane (17), ␥-chymotrypsin in hexane (18,19), and elastase in acetonitrile (20), have been elucidated and found to be essentially the same as in water. Furthermore, the structures in hexane of a ␥-chymotrypsin-product complex has been determined (19) and that of a tetrahedral complex claimed (18). However, to provide further insight into the enzyme mechanism in organic solvents, structures of covalent reaction intermediates should be determined. To the best of our knowledge, no such structures have been available, until now.All the aforementioned enzyme structures are of serine proteases, for which the universal reaction intermediate in water is an acyl-enzyme. In organic solvents, however, the formation of such an intermediate in the catalysis by subtilisin is supported by kinetic but not direct structural data (21-24).Moreover, it is simply presumed that the structure of the enzyme-substrate intermediate formed in different solvents is the same (9, 10). The most direct way to experimentally test this assumpti...

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Section: Resultssupporting

confidence: 84%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…In organic solvents, however, the formation of such an intermediate in the catalysis by subtilisin is supported by kinetic but not direct structural data (21)(22)(23)(24).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Schmitke

Stern

Klibanov

1998

Proc. Natl. Acad. Sci. U.S.A.

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“…enzyme activity, specificity and enantioselectivity) in conventional solvents, is susceptible to solvent properties including dielectric constant, partition coefficient and hydrophobicity. [85][86][87][88][89][90][91] Therefore, it is not an unexpected result that changes in the properties of SCFs dramatically affect enzyme activity.…”

Section: Methodsmentioning

confidence: 99%

Recent trends in whole cell and isolated enzymes in enantioselective synthesis

Milner¹,

Maguire²

2012

Arkivoc

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Modern synthetic organic chemistry has experienced an enormous growth in biocatalytic methodologies; enzymatic transformations and whole cell bioconversions have become generally accepted synthetic tools for asymmetric synthesis. This review details an overview of the latest achievements in biocatalytic methodologies for the synthesis of enantiopure compounds with a particular focus on chemoenzymatic synthesis in non-aqueous media, immobilisation technology and dynamic kinetic resolution. Furthermore, recent advances in ketoreductase technology and their applications are also presented.Keywords: Biocatalysis, kinetic and dynamic kinetic resolution, Baker's yeast, ketoreductases General IntroductionAsymmetric synthesis is the preferential formation of one stereoisomer of a chiral target compound to another; when scientists at GlaxoSmithKline, AstraZeneca and Pfizer 1 examined 128 syntheses from their companies, they found as many as half of the drug compounds made by their process research and development groups are not only chiral but also contain an average of two chiral centres each. 2In 2006 just 25 % were derived from the chiral pool and over 50 % employed chiral technologies. 1In order to meet regulatory requirements enantiomeric purities of 99.5 % were deemed necessary by the FDA. 3 This is one of the biggest challenges which face chemists today, primarily due to the recognition of the fact that different enantiomers of the same compound can interact differently in biological systems. As a consequence, the production of single enantiomers instead of racemic mixtures has become an important process in the pharmaceutical and agrochemical industry. Several routes can lead to the desired enantiomer including transition metal-, [4][5][6] organo-5,7-10 and bio-catalysis [11][12][13][14][15] and these have been thoroughly reviewed in the literature. 16,17 2. Biocatalysis IntroductionBiocatalysis involves the use of enzymes or whole cells (containing the desired enzyme or enzyme system) as catalysts for chemical reactions. A timeline of historic events in biocatalysis and biotechnology is outlined in Table 1. [18][19][20] Reviews and Accounts Novel methodologies for discovering industrial enzymes based on genomic sequencing and phage display (discussed further in Section 1.3), 21,22 as well as highly effective optimisation tools based on chemical, physical and molecular biology approaches, 23 have improved the access to biocatalysts, increased their stability, and radically broadened their specificity. 24This greater availability of catalysts with superior qualities including the use of new bioengineering tools contributed significantly to the development of new industrial processes. Biocatalytic methodologies for organic synthesis were outlined by Woodley et al. in three categories: established, emerging and expanding chemistries as depicted in Figure 1. 25 Enzyme classesBy the late 1950s it had become evident that the nomenclature of enzymes without any guiding authority, in a period when the...

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Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions

Zheng¹,

Ornstein²

1996

Biopolymers

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Kinetic isotope effect investigation of enzyme mechanisms in organic solvents

Cited by 29 publications

References 1 publication

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

Recent trends in whole cell and isolated enzymes in enantioselective synthesis

Molecular dynamics of subtilisin Carlsberg in aqueous and nonaqueous solutions

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