m-Fluorotyrosine substitution in beta-galactosidase; evidence for the existence of a catalytically active tyrosine.
The pH profiles of beta-galactosidase, having tyr replaced by m-fluorotyrosine, were compared to those of normal enzyme. The inflection point on the alkaline side was lowered about 1.5 pH units in the fluoro-enzyme, corresponding to the difference in the phenolic pKa values of m-fluorotyrosine and tyr. When glycosidic bond breakage was rate-limiting, the Vm at pH 7.0 was higher for the fluoro-enzyme. When hydrolysis was rate-limiting or when acceptors which made transgalactosylis rate-limiting were used, the Vm was lower for the fluoro-enzyme. This shows that a tyr in beta-galactosidase is a general-acid catalyst in the glycosidic bond breaking reaction and a tyr (probably the same one) is a general-base catalyst in the hydrolytic reaction.
D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology; D005690:Galactose / Q000378:metabolism; D005696:Galactosidases / Q000378:metabolism*; D005697:Galactosides / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009598:Nitrophenylgalactosides / Q000378:metabolism; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000031:analogs & derivatives*; D014993:Xylitol / Q000378:metabolism; D014994:Xylose / Q000378:metabolism; D001616:beta-Galactosidase / Q000378:metabolism*
D005697:Galactosides; C001245:3-fluorotyrosine; D009598:Nitrophenylgalactosides; D014443:Tyrosine; C046399:phenyl-D-galactopyranoside; D014994:Xylose; D005696:Galactosidases; D001616:beta-Galactosidase; D014993:Xylitol; D005690:Galactose