Ethylenediamine and aminoacetonitrile catalyzed decarboxylation of oxalacetate

Leussing, D. L.; Raghavan, N. V.

doi:10.1021/ja00537a039

Cited by 33 publications

(27 citation statements)

References 1 publication

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“…Conversely, since the imine mechanism involves hydrolysis of the immonium intermediate by water, 18 O labeling should be observed in the C2 carbonyl group. The reaction was carried out in buffered H 2 16 O and H 2 18 O, monitored by UV to completion, and analyzed by ESI-MS. Only unlabeled product (158 Da) was observed with both enzymes with no indication of the 18 Olabeled compound (160 Da) within the detection limits (< 2 %). These results further support the conclusion that the acid/base mechanism is maintained in wild-type enzymes as well as in the P1A mutant.…”

Section: Resultsmentioning

confidence: 99%

Mutants of 4-Oxalocrotonate Tautomerase Catalyze the Decarboxylation of Oxaloacetate through an Imine Mechanism

et al. 2002

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Section: Resultsmentioning

confidence: 99%

Mutants of 4-Oxalocrotonate Tautomerase Catalyze the Decarboxylation of Oxaloacetate through an Imine Mechanism

et al. 2002

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“…Arg 292 was substituted with glutamate to introduce a negative charge that might destabilize the ␤-carboxylate group (29,30) and thus enhance ␤-decarboxylation. If the ratio of ␤-decarboxylase to transaminase activity is considered rather than the absolute k cat value, AspAT Y225R/R292E/R386A is indeed a more specific L-aspartate ␤-decarboxylase than its counterpart with the R292K substitution, its ␤-decarboxylase activity being 100 times higher than its transaminase activity (Table I).…”

Section: Resultsmentioning

confidence: 99%

Conversion of Aspartate Aminotransferase into anl-Aspartate β-Decarboxylase by a Triple Active-site Mutation

Graber

Kasper

Malashkevich

et al. 1999

Journal of Biological Chemistry

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The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate ␤-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg 386 interacts with the ␣-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr 225 is in its vicinity, forming a hydrogen bond with O-3 of the cofactor; and Arg 292 interacts with the distal carboxylate group of the substrate. In the triplemutant enzyme, k cat for ␤-decarboxylation of L-aspartate was 0.08 s ؊1 , whereas k cat for transamination was decreased to 0.01 s ؊1. AspAT was thus converted into an L-aspartate ␤-decarboxylase that catalyzes transamination as a side reaction. The major pathway of ␤-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single-or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low ␤-decarboxylase activity. The arginine shift mutation Y225R/ R386A elicits ␤-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.In the engineering of protein catalysts with new functional properties, the modification of existing enzymes provides an alternative to the production of catalytic antibodies or, in a more distant future, the de novo design of enzymes. Enzyme engineering may be expected to contribute to elucidating both the structural basis of the functional properties and the course of the molecular evolution. Several attempts to change the substrate specificity of an enzyme by substitution of the substrate-binding residues have succeeded (Refs. 1-9; for a review, see Ref. 6). Among the pyridoxal 5Ј-phosphate-dependent enzymes, aspartate aminotransferase (AspAT) 1 has been converted by multiple active-site mutations into an L-tyrosine aminotransferase (5) and by directed molecular evolution into an L-branched-chain amino acid aminotransferase (7,8). Tyrosine phenol-lyase has been engineered by a double mutation to act as a dicarboxylic-acid ␤-lyase (an enzyme not found in nature) that degrades aspartate to pyruvate, ammonia, and formate (9). However, as yet, no change in the reaction specificity of an enzyme has been reported, with the exception of the conversion of papain into a peptide-nitrile hydratase (10). A change in the reaction specificity may be claimed if a new catalytic activity not inherent in the wild-type enzyme is generated and the original activity of the wil...

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“…One example of introducing catalytic machinery to promote a different reaction is provided by the generation of oxaloacetate decarboxylase activity in 4‐oxalocrotonate tautomerase (4‐OT). In small‐molecule45 or peptide model systems,46 catalysis of oxaloacetate decarboxylation simply requires formation of a Schiff base between the substrate and a primary amine (Scheme ). The enzyme 4‐OT does not normally form a covalent intermediate with its substrate, but rather uses the secondary amine of Pro1 as an acid/base to effect a proton shift 47.…”

Section: Strategies For Active‐site Redesignmentioning

confidence: 99%

Minimalist Active‐Site Redesign: Teaching Old Enzymes New Tricks

2007

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Although nature evolves its catalysts over millions of years, enzyme engineers try to do it a bit faster. Enzyme active sites provide highly optimized microenvironments for the catalysis of biologically useful chemical transformations. Consequently, changes at these centers can have large effects on enzyme activity. The prediction and control of these effects provides a promising way to access new functions. The development of methods and strategies to explore the untapped catalytic potential of natural enzyme scaffolds has been pushed by the increasing demand for industrial biocatalysts. This Review describes the use of minimal modifications at enzyme active sites to expand their catalytic repertoires, including targeted mutagenesis and the addition of new reactive functionalities. Often, a novel activity can be obtained with only a single point mutation. The many successful examples of active-site engineering through minimal mutations give useful insights into enzyme evolution and open new avenues in biocatalyst research.

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Ethylenediamine and aminoacetonitrile catalyzed decarboxylation of oxalacetate

Cited by 33 publications

References 1 publication

Mutants of 4-Oxalocrotonate Tautomerase Catalyze the Decarboxylation of Oxaloacetate through an Imine Mechanism

Mutants of 4-Oxalocrotonate Tautomerase Catalyze the Decarboxylation of Oxaloacetate through an Imine Mechanism

Conversion of Aspartate Aminotransferase into anl-Aspartate β-Decarboxylase by a Triple Active-site Mutation

Minimalist Active‐Site Redesign: Teaching Old Enzymes New Tricks

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