Stereochemistry and Mechanism of Reactions Catalyzed by Tyrosine Phenol-Lyase from Escherichia intermedia

Abstract

Stereochemical studies on tyrosine phenol-lyase from Escherichia intermedia have shown that the α,β-elimination reactions of ʟ-serine and ᴅ- and ʟ-tyrosine proceed with retention of config­uration at C-β. Stereospecifically (β-tritiated ʟ-serine is slowly racemized at C-β Deuterium from the α-position of ʟ-tyrosine is partially transferred to C-4 of the phenol formed when the α,β- elimination reaction is carried out in H2O, although no transfer of α-1H in 2H2O was seen. The result favors tautomerization of the p-hydroxyphenyl to a cyclohexadienonyl moiety prior to carbon-carbon bond cleavage. In the conversion of ʟ- to ᴅ-alanine catalyzed by tyrosine phenol- lyase, some a-hydrogen recycling is observed, pointing to a single-base racemization mechanism. Attempts to demonstrate cofactor motion during racemization by NaBH4 reduction of [3H]PLP- enzyme: ᴅ- and ʟ-alanine complexes failed, but showed that, as in other PLP enzymes, the holoenzyme is reduced preferentially from the Re face with respect to C-4′ of PLP and enzyme- substrate complexes preferentially from the Si face.