New developments in enzymatic peptide synthesis

Wong, Chi Huey; Wang, K. T.

doi:10.1007/bf01918376

Cited by 68 publications

(21 citation statements)

References 37 publications

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“…Enzyme catalysis in nonaqueous solvents has attracted a lot of attention recently due to its nonconventional approach and potential applications in bioorganic synthesis (Barbas and Wong, 1987;Laane, 1987;Arnold, 1988Arnold, , 1990Kitaguchi and Klibanov, 1989;Kuhl et aL, 1990;Phillips et al, 1990;Wong et aL, 1990;Wong and Wong, 1991;Zhong et al, 1991;Nagashimh et al, 1992). Enzymes of different sources, including alcalase, which can maintain their enzymatic activities to various extents in organic solvents have also been documented (Schulze and Klibanov, 1991).…”

Section: Discussionmentioning

confidence: 99%

Physicochemical properties of alkaline serine proteases in alcohol

Chen

et al. 1995

J Protein Chem

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The alkaline proteases subtilisin Carlsberg and alcalase possess substantial enzymatic activity even when dissolved in ethanol. The crude enzymes were purified by gel filtration and the main fractions suspended in ethanol to give a translucent suspension. Both the supernatant and the resuspended precipitate after high-speed centrifugation were found to have enzymatic activities. The solubility of subtilisin Carlsberg in anhydrous ethanol was found to be 45.1 micrograms/ml and that of alcalase was 48.1 micrograms/ml by Coomassie blue dye-binding method using bovine serum albumin as a standard. In the presence of water, the solubility of both enzymes increased with water content. The stability of enzymes incubated in ethanol was assayed by their amidase and transesterase activities using Ala-Ala-Pro-Phe-pNA as substrate in phosphate buffer (pH8.2) and Moz-Leu-OBzl as substrate in anhydrous ethanol, respectively. The soluble enzymes have a half-life of about 36 hr and that of suspended enzymes about 50 hr in the amidase activity assay, whereas the same soluble enzymes have a half-life of about several hours and that of suspended enzymes 1 h by the transesterase activity assay. The stability of both enzymes decreased as water concentration increased. The diastereoselectivity of the enzyme-catalyzed hydrolysis of diastereo pairs of tetrapeptide esters, L-Ala-L-Ala-(D- or L-)Pro-L-Phe-OMe and L-Ala-L-Ala-(D- or L-)Ala-L-Phe-OMe, in phosphate is as high as that of the transesterification of these substrates in ethanol. It is concluded that active sites and selectivity of alkaline serine proteases in anhydrous alcohol are probably very similar to those in aqueous solution in spite of the fact that a lower reactivity is usually associated with the enzymes in nonaqueous solvents.

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Section: Discussionmentioning

confidence: 99%

Physicochemical properties of alkaline serine proteases in alcohol

Chen

et al. 1995

J Protein Chem

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“…The kcat of PC3 actually increases in 20% DMF, and its Km is much less sensitive to the organic solvent (7- (7). Subtilisin can also acylate sugars (18) and steroids (8) regioselectively and will catalyze peptide synthesis either by direct reversal of the hydrolytic process or by aminolysis of N-protected amino acid or peptide esters (19).…”

Section: Figmentioning

confidence: 99%

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.

Chen

Arnold

1993

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

484

235

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Random mutagenesis has been used to engineer the protease subtilisin E to function in a highly nonnatural environment-high concentrations of a polar organic solvent. Sequential rounds of mutagenesis and screening have yielded a variant (PC3) that hydrolyzes a peptide substrate 256 times more efficiently than wild-type subtiisin in 60% dimethylformamide. PC3 subtilisin E and other variants containing different combinations of amino acid substitutions are effective catalysts for transesterification and peptide synthesis in dimethylformamide and other organic media. Starting with a variant containing four effective amino acid substitutions (D60N, D97G, Q103R, and N218S; where, for example, D60N represents Asp-60 -) Asn), six additional mutations (G131D, E156G, N181S, S182G, S188P, and T255A) were generated during three sequential rounds of mutagenesis and screening. The 10 substitutions are clustered on one face of the enzyme, near the active site and substrate binding pocket, and all are located in loops that connect core secondary structure elements and exhibit considerable sequence variability in subtilisins from different sources. These variable surface loops are effective handles for "tuning" the activity of subtilisin. Seven of the 10 amino acid substitutions in PC3 are found in other natural subtilisins. Great variability is exhibited among naturally occurring sequences that code for similar three-dimensional structures-it is possible to make use of this sequence flexibility to engineer enzymes to exhibit features not previously developed (or required) for function in vivo.With exquisite substrate specificities and high reaction selectivities, enzymes offer tremendous advantages for chemical synthesis. Nonetheless, practical applications of enzyme catalysis have been limited, due in large part to relatively poor stabilities and catalytic activities under the conditions that characterize industrial processes: high temperatures, extremes of pH, or nonaqueous solvents. Enzymes evolved for the survival benefit of an organism may not exhibit features essential for in vitro application.A large body of experience in tailoring enzyme properties by making selected substitutions of the enzyme's amino acid sequence has accumulated. A "rational design" approach involving site-directed mutagenesis is inefficient, however, in the absence of detailed structural information or when the molecular basis for the property of interest is poorly understood. In such cases, random mutagenesis combined with selection or screening can be a useful alternative for generating both the desired improvements and a data base for future rational approaches to protein design. Random mutagenesis has been used to enhance or alter various enzyme features, including thermal stability (1-3), alkaline stability (4), and substrate specificity (5), and to recover the catalytic The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U...

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“…However, catalysis of peptide bond formation between native protein0peptide segments by proteases in neat aqueous solution usually do not occur appreciably even in favorable situations entailing stereochemical proximity of the reacting ends, as in proteolytically nicked and thermodynamically stable noncovalent complexes of proteins, primarily because the protease-catalyzed peptide bond hydrolytic equilibria is not adequately shifted in the direction of ligation in 55.5 M water. Nonetheless, proteases catalyze the condensation of peptides in aqueous solution with the use of C-terminal active esters~Schellenberger & Jakubke, 1991; Wong & Wang, 1991;Braisted et al, 1997!. Here we report that generation of productdirected conformational traps can act as a driving force for the protease-catalyzed splicing of native peptide segments in neat aqueous solutions, thereby obviating the necessity of active esters or organic cosolvents or strong interaction between the reacting peptides.…”

Section: Discussionmentioning

confidence: 87%

An enigmatic peptide ligation reaction: Protease‐catalyzed oligomerization of a native protein segment in neat aqueous solution

2000

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We report an enigmatic peptide ligation reaction catalyzed by Glu-specific Staphylococcus aureus V8 protease that occurs in neat aqueous solution around neutral pH utilizing a totally unprotected peptide substrate containing free a-carboxyl and a-amino groups. V8 protease catalyzed a chain of ligation steps between pH 6 and 8 at 4 8C, producing a gamut of covalent oligomers~dimer through octamer or higher! of a native protein segment TAAAKFE~S39! derived from ribonuclease A~RNAse A!. Size-exclusion chromatography suggested the absence of strong interaction between the reacting peptides. The circular dichroism spectra of monomer through pentamer showed length-dependent enhancement of secondary structure in the oligomers, suggesting that protease-catalyzed ligation of a monomer to an oligomer resulted in a product that was more structured than its precursor. The relative conformational stability of the oligomers was reflected in their ability to resist proteolysis, indicating that the oligomerization reaction was facilitated as a consequence of the "conformational trapping" of the product. The ligation reaction proceeded in two phases-slow formation and accumulation of the dimer followed by a fast phase of oligomerization, implying that the conformational trap encountered in the oligomerization reaction was a two-step process. The Gly substitution at any position of the TAAAKFE sequence was deleterious, suggesting that the first step of the conformational trap, namely the dimerization reaction, that proceeded very slowly even with the parent peptide, was quite sensitive to amino acid sequence. In contrast, the oligomerization reaction of an Ala analog, AAAAKFE, occurred in much the same way as S39, albeit with faster rate, suggesting that Ala substitution stabilized the overall conformational trapping process. The results suggest the viability of the product-directed "conformational trap" as a mechanism to achieve peptide ligation of totally unprotected peptide fragments in neat aqueous solution. Further, the study projects the presence of considerable innate synthetic potential in V8 protease, baring rich possibilities of protein engineering of this enzyme to generate a "V8 peptide ligase."

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New developments in enzymatic peptide synthesis

Cited by 68 publications

References 37 publications

Physicochemical properties of alkaline serine proteases in alcohol

Physicochemical properties of alkaline serine proteases in alcohol

Tuning the activity of an enzyme for unusual environments: sequential random mutagenesis of subtilisin E for catalysis in dimethylformamide.

An enigmatic peptide ligation reaction: Protease‐catalyzed oligomerization of a native protein segment in neat aqueous solution

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