Assessing the Deamination Rate of a Covalent Aminomutase Adduct by Burst Phase Analysis

Burst-phase kinetic analysis was used to evaluate the deamination rate of the aminated–methylidene imidazolone (NH₂–MIO) adduct of a <i>Taxus</i> phenylalanine aminomutase. The kinetic parameters were interrogated by a non-natural substrate (<i>S</i>)-styryl-α-alanine that yielded a chromophoric styrylacrylate product upon deamination by the aminomutase. Transient inactivation of the enzyme by the NH₂–MIO adduct intermediate resulted in an initial burst of product, with reactivation by deamination of the adduct. This study validated the rate constants of a kinetic model demonstrating that the NH₂–MIO adduct and cinnamate intermediate are sufficiently retained to catalyze the natural α- to β-phenylalanine isomerization

A Bacterial Tyrosine Aminomutase Proceeds through Retention or Inversion of Stereochemistry To Catalyze Its Isomerization Reaction

β-Amino acids are biologically active compounds of interest in medicinal chemistry. A class I lyase-like family of aminomutases isomerizes (<i>S</i>)-α-arylalanines to the corresponding β-amino acids by exchange of the NH₂/H pair. This family uses a 3,5-dihydro-5-methylidene-4<i>H</i>-imidazol-4-one (MIO) group within the active site to initiate the reaction. The absolute stereochemistry of the product is known for an MIO-dependent tyrosine aminomutase from Chondromyces crocatus (<i>Cc</i>TAM) that isomerizes (<i>S</i>)-α-tyrosine to (<i>R</i>)-β-tyrosine. To evaluate the cryptic stereochemistry of the <i>Cc</i>TAM mechanism, (2<i>S</i>,3<i>S</i>)-[2,3-²H₂]- and (2<i>S</i>,3<i>R</i>)-[3-²H]-α-tyrosine were stereoselectively synthesized from unlabeled (or [²H]-labeled) (4′-hydroxyphenyl)­acrylic acids by reduction with D₂ (or H₂) gas and a chiral Rh-Prophos catalyst. GC/EIMS analysis of the [²H]-β-tyrosine biosynthesized by <i>Cc</i>TAM revealed that the α-amino group was transferred to C_β of the phenylpropanoid skeleton with retention of configuration. These labeled substrates also showed that the <i>pro</i>-(3<i>S</i>) proton exchanges with protons from the bulk media during its migration to C_α during catalysis. ¹H- and ²H NMR analyses of the [²H]-β-tyrosine derived from (2<i>S</i>)-[3,3-²H₂]-α-tyrosine by <i>Cc</i>TAM catalysis showed that the migratory proton attached to C_α of the product also with retention of configuration. <i>Cc</i>TAM is stereoselective for (<i>R</i>)-β-tyrosine (85%) yet also forms the (<i>S</i>)-β-tyrosine enantiomer (15%) through inversion of configuration at both migration termini, as described herein. The proportion of the (<i>S</i>)-β-isomer made by <i>Cc</i>TAM during steady state interestingly increased with solvent pH, and this effect on the proposed reaction mechanism is also discussed

A Bacterial Tyrosine Aminomutase Proceeds through Retention or Inversion of Stereochemistry To Catalyze Its Isomerization Reaction

β-Amino acids are biologically active compounds of interest in medicinal chemistry. A class I lyase-like family of aminomutases isomerizes (<i>S</i>)-α-arylalanines to the corresponding β-amino acids by exchange of the NH₂/H pair. This family uses a 3,5-dihydro-5-methylidene-4<i>H</i>-imidazol-4-one (MIO) group within the active site to initiate the reaction. The absolute stereochemistry of the product is known for an MIO-dependent tyrosine aminomutase from Chondromyces crocatus (<i>Cc</i>TAM) that isomerizes (<i>S</i>)-α-tyrosine to (<i>R</i>)-β-tyrosine. To evaluate the cryptic stereochemistry of the <i>Cc</i>TAM mechanism, (2<i>S</i>,3<i>S</i>)-[2,3-²H₂]- and (2<i>S</i>,3<i>R</i>)-[3-²H]-α-tyrosine were stereoselectively synthesized from unlabeled (or [²H]-labeled) (4′-hydroxyphenyl)­acrylic acids by reduction with D₂ (or H₂) gas and a chiral Rh-Prophos catalyst. GC/EIMS analysis of the [²H]-β-tyrosine biosynthesized by <i>Cc</i>TAM revealed that the α-amino group was transferred to C_β of the phenylpropanoid skeleton with retention of configuration. These labeled substrates also showed that the <i>pro</i>-(3<i>S</i>) proton exchanges with protons from the bulk media during its migration to C_α during catalysis. ¹H- and ²H NMR analyses of the [²H]-β-tyrosine derived from (2<i>S</i>)-[3,3-²H₂]-α-tyrosine by <i>Cc</i>TAM catalysis showed that the migratory proton attached to C_α of the product also with retention of configuration. <i>Cc</i>TAM is stereoselective for (<i>R</i>)-β-tyrosine (85%) yet also forms the (<i>S</i>)-β-tyrosine enantiomer (15%) through inversion of configuration at both migration termini, as described herein. The proportion of the (<i>S</i>)-β-isomer made by <i>Cc</i>TAM during steady state interestingly increased with solvent pH, and this effect on the proposed reaction mechanism is also discussed