Stereochemistry and mechanism of reactions catalyzed by tyrosine phenol-lyase from Escherichia intermedia.

Stereochemical studies on tyrosine phenol-lyase from Escherichia intermedia have shown that the alpha, beta-elimination reactions of L-serine and D- and L-tyrosine proceed with retention of configuration at C-beta. Stereospecifically beta-tritiated L-serine is slowly racemized at C-beta. Deuterium from the alpha-position of L-tyrosine is partially transferred to C-4 of the phenol formed when the alpha, beta-elimination reaction is carried out in H2O, although no transfer of alpha-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 L- to D-alanine catalyzed by tyrosine phenol-lyase, some alpha-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: D- and L-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.