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.
Brooks
B
B
Department of Biological Chemistry, School of Medicine, University of California, Davis 95616, USA.
Phillips
R S
RS
Benisek
W F
WF
D019943:Amino Acid Substitution / Q000235:genetics; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D005462:Fluorine Radioisotopes / Q000378:metabolism; D016946:Gram-Negative Aerobic Rods and Cocci / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006878:Hydroxides / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D010649:Phenylalanine / Q000235:genetics; D013251:Steroid Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism*
D005462:Fluorine Radioisotopes; D006878:Hydroxides; C001245:3-fluorotyrosine; D014443:Tyrosine; D010649:Phenylalanine; C060489:2-fluorotyrosine; C031356:hydroxide ion; D013251:Steroid Isomerases; C022660:steroid delta-isomerase