Biochemical analysis reveals stereospecific catalysis by the H297N mutant in mandelate racemase, indicating His 297 serves as the stereospecific general base for (R)-mandelate.
Key Points
Determine the functional role of His 297 in mandelate racemase by characterizing the structure and stereospecific catalytic activity of the H297N mutant.
Substituted His 297 with asparagine using site-directed mutagenesis and determined the crystal structure at 2.2-Å resolution via molecular replacement.
Assayed catalytic elimination of bromide from racemic p-(bromomethyl)mandelate and solvent deuterium exchange into mandelate enantiomers across varied pD conditions.
The H297N mutant had no detectable racemase activity and showed no structural alterations relative to the wild-type enzyme at 2.2-Å resolution.
H297N catalyzed bromide elimination from racemic substrate in 45% yield at wild-type rates and mediated alpha-proton exchange solely on (S)-mandelate at a 3.3-fold reduced rate, reflecting a Lys 166 pKa of 6.4 in D2O.