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...

The electron distribution in the coenzyme-substrate adduct of aspartate aminotransferase was changed by replacing active-site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates L-aspartate to L-alanine (kcat = 0.04 s-1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat = 0.19 s-1). Molecular-dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal-5'-phosphate- aspartate adduct and an altered electrostatic potential around its beta-carboxylate group underlie the 650,000-fold increase in the ratio of beta-decarboxylase/transaminase activity.

We describe a methodology to calculate the relative free energies of protein-peptide complex formation. The interaction energy was decomposed into nonpolar, electrostatic and entropic contributions. A free energy-surface area relationship served to calculate the nonpolar free energy term. The electrostatic free energy was calculated with the finite difference Poisson-Boltzmann method and the entropic contribution was estimated from the loss in the conformational entropy of the peptide side chains. We applied this methodology to a series of DnaK*peptide complexes. On the basis of the single known crystal structure of the peptide-binding domain of DnaK with a bound heptapeptide, we modeled ten other DnaK*heptapeptide complexes with experimentally measured K(d) values from 0.06 microM to 11 microM, using molecular dynamics to refine the structures of the complexes. Molecular dynamic trajectories, after equilibration, were used for calculating the energies with greater accuracy. The calculated relative binding free energies were compared with the experimentally determined free energies. Linear scaling of the calculated terms was applied to fit them to the experimental values. The calculated binding free energies were between -7.1 kcal/mol and - 9.4 kcal/mol with a correlation coefficient of 0.86. The calculated nonpolar contributions are mainly due to the central hydrophobic binding pocket of DnaK for three amino acid residues. Negative electrostatic fields generated by the protein increase the binding affinity for basic residues flanking the hydrophobic core of the peptide ligand. Analysis of the individual energy contributions indicated that the nonpolar contributions are predominant compared to the other energy terms even for peptides with low affinity and that inclusion of the change in conformational entropy of the peptide side chains does not improve the discriminative power of the calculation. The method seems to be useful for predicting relative binding energies of peptide ligands of DnaK and might be applicable to other protein-peptide systems, particularly if only the structure of one protein-ligand complex is available.

Tyrosine phenol-lyase (TPL), which catalyzes the ␤-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the ␣-family of vitamin B 6 -dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/ V283R of TPL increased the ␤-elimination activity toward dicarboxylic amino acids at least 10 4 -fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (k cat ‫؍‬ 0.21 s ؊1 ) was two times higher than that toward L-tyrosine. ␤-Elimination and transamination as a minor side reaction (k cat ‫؍‬ 0.001 s ؊1 ) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid ␤-lyase is an enzyme not found in nature.The pyridoxal 5Ј-phosphate-dependent enzymes (B 6 enzymes) catalyze a wide variety of reactions in the metabolism of amino acids (1). A comparison of amino acid sequences has shown that the majority of B 6 enzymes belong to the large ␣/␥-superfamily of homologous B 6 enzymes (2, 3). Tyrosine phenol-lyase (TPL) 1 of Citrobacter freundii is a member of the ␣-family. It catalyzes the ␤-elimination of L-tyrosine to produce phenol, pyruvate, and ammonium (Equation 1).A number of amino acids with suitable leaving groups on C␤, such as L-serine and O-acyl-L-serines (4), L-cysteine, S-alkyl-Lcysteines (4, 5), and 3-chloro-L-alanine, are also substrates for ␤-elimination. Moreover, TPL has been found to catalyze markedly slower side reactions, i.e. ␤-replacement reactions (6, 7), racemization of alanine (8, 9), as well as transamination reactions of its substrates L-tyrosine, L-serine, and of the competitive inhibitors L-alanine, L-phenylalanine, and L-m-tyrosine (10).X-ray crystallographic structure analysis has shown the folding pattern of the polypeptide chain of tetrameric TPL from C. freundii to be similar to that of dimeric aspartate aminotransferase (AspAT) (11), which, like TPL, is a member of the ␣-family of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes. Despite their similarity in secondary and tertiary structure, the two enzymes show only low sequence identity, e.g. 23% between TPL of C. freundii and AspAT of Escherichia coli. AspAT catalyzes the reversible transaminatio...

The electron distribution in the coenzyme‐substrate adduct of aspartate aminotransferase was changed by replacing active‐site Arg386 with alanine and introducing a new arginine residue nearby. [Y225R, R386A]Aspartate aminotransferase decarboxylates l‐aspartate to l‐alanine (kcat= 0.04 s−1), while its transaminase activity towards dicarboxylic amino acids is decreased by three orders of magnitude (kcat= 0.19 s−1). Molecular‐dynamics simulations based on the crystal structure of the mutant enzyme suggest that a new hydrogen bond to the imine N atom of the pyridoxal‐5′‐phosphate‐aspartate adduct and an altered electrostatic potential around its β‐carboxylate group underlie the 650000‐fold increase in the ratio of β‐decarboxylase/transaminase activity.