High-efficiency incorporation in vivo of tyrosine analogues with altered hydroxyl acidity in place of the catalytic tyrosine-14 of Delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: effects of the modifications on isomerase kinetics.

Versions of the Y55F/Y88F modified form of Delta 5-3-ketosteroid isomerase in which the active-site tyrosine-14 is replaced by 2-fluorotyrosine, 3-fluorotyrosine, and 2,3-difluorotyrosine, amino acids having progressively greater acidity of their phenolic hydroxyls, have been expressed in an Escherichia coli host and purified to high homogeneity. The steady-state kinetic properties of Y55F/Y88F KSI and its fluorotyrosine modified forms have been determined. The mechanistic implications of the results are presented and discussed.