▁ ▁The ▁rate - det erm ining ▁step ( R DS ) ▁in ▁this ▁mechanism ▁is ▁related ▁to ▁formation ▁of ▁the ▁ox o - car ben ium ▁intermediate . ▁There ▁were ▁some ▁contradict ory ▁results ▁to ▁indicate ▁the ▁exact ▁R DS . ▁By ▁trac ing ▁the ▁formation ▁of ▁product ▁( p - nit ro phen ol ), ▁it ▁was ▁discovered ▁that ▁the ▁R DS ▁can ▁change ▁over ▁different ▁temper atures , ▁which ▁was ▁a ▁reason ▁for ▁those ▁contradict ory ▁results . ▁At ▁a ▁higher ▁temperature ▁the ▁R DS ▁is ▁formation ▁of ▁g ly cos yl ▁en zym e ▁intermediate ▁and ▁at ▁a ▁lower ▁temperature ▁the ▁break ▁down ▁of ▁that ▁intermediate . ▁ ▁K osh land ▁▁ ▁In ▁an ▁early ▁debate ▁in ▁ 1 9 6 9 , ▁D ahl qu ist ▁proposed ▁a ▁c oval ent ▁mechanism ▁for ▁ly so zym e ▁based ▁on ▁kin etic ▁is ot ope ▁effect , ▁but ▁for ▁a ▁long ▁time ▁the ▁ion ic ▁mechanism ▁was ▁more ▁accepted . ▁In ▁ 2 0 0 1 , ▁a ▁re vised ▁mechanism ▁was ▁proposed ▁by ▁V oc ad lo ▁via ▁a ▁c oval ent ▁but ▁not ▁ion ic ▁intermediate . ▁Ev idence ▁from ▁E SI - MS ▁analysis ▁ ▁indicated ▁a ▁c oval ent ▁intermediate . ▁A ▁ 2 - flu oro ▁subst it uted ▁substr ate ▁was ▁used ▁to ▁lower ▁the ▁reaction ▁rate ▁and ▁accum ulate ▁an ▁intermediate ▁for ▁character ization . ▁The ▁am ino ▁acid ▁side - ch ains ▁gl ut am ic ▁acid ▁ 3 5 ▁(