- fold , ▁respectively , ▁imp lic ating ▁Asp - 9 9 ▁as ▁important ▁for ▁en zym atic ▁activity . ▁Wu ▁et ▁al . ▁proposed ▁a ▁mechanism ▁that ▁involves ▁both ▁T yr - 1 4 ▁and ▁Asp - 9 9 ▁forming ▁hydro gen ▁b onds ▁directly ▁to ▁O - 3 ▁of ▁the ▁ster oid . ▁This ▁mechanism ▁was ▁challeng ed ▁by ▁Z ha o ▁et ▁al ., ▁who ▁post ulated ▁a ▁hydro gen ▁bond ing ▁network ▁with ▁Asp - 9 9 ▁hydro gen ▁bond ing ▁to ▁T yr - 1 4 , ▁which ▁in ▁turn ▁forms ▁a ▁hydro gen ▁bond ▁to ▁O - 3 . ▁ ▁More ▁recently , ▁the ▁H ersch lag ▁group ▁util ized ▁un natural ▁am ino ▁acid ▁incorpor ation ▁to ▁ass ay ▁the ▁importance ▁of ▁T yr - 1 4 ▁to ▁K SI ▁catal ysis . ▁ ▁The ▁natural ▁ty ros ine ▁resid ue ▁was ▁subst it uted ▁with ▁un natural ▁hal ogen ated ▁am ino ▁ac ids ▁surve ying ▁a ▁range ▁of ▁p K a ' s . ▁ ▁There ▁was ▁very ▁little ▁difference ▁in ▁K SI ▁catal yt ic ▁turn over ▁with ▁decre asing ▁p K a , ▁suggesting , ▁in ▁contrast ▁to ▁the ▁electro static ▁studies ▁out lined ▁above , ▁that ▁o xy an ion ▁hole ▁stabil ization ▁is ▁not ▁primarily ▁important ▁for ▁catal ysis . ▁ ▁Asp - 3 8 ▁general ▁acid ic / basic ▁activity ▁and ▁effective ▁mol ar ity ▁was ▁prob ed ▁by ▁the ▁H ersch lag ▁group ▁through ▁site - direct ed