Chondroitin AC lya
se from
Flavobacterium heparinum degrade
s chondroitin
sulfate glyco
saminoglycan
s via an elimination mechani
sm re
sulting in di
saccharide
s or oligo
saccharide
s with
s/gifchar
s/Delta.gif" BORDER=0 >4,5-un
saturateduronic acid re
sidue
s at their nonreducing end. Mechani
stic detail
s concerning the ordering of the bond-breaking and -forming
step
s of thi
s enzymatic reaction are nonexi
stent, mainly due to the inhomogeneou
snature of the polymeric
sub
strate
s. The creation of a new cla
ss of
synthetic
sub
strate
s for thi
s enzyme ha
sallowed the mea
surement of defined and reproducible
kcat and
Km value
s and ha
s expanded the range ofmechani
stic
studie
s that can be performed. The primary deuterium kinetic i
sotope effect upon
kcat/
Km forthe ab
straction of the proton
s/gifchar
s/alpha.gif" BORDER=0> to the carboxylic acid wa
s mea
sured to be 1.67 ± 0.07,
showing thatdeprotonation occur
s in a rate-limiting
step. U
sing
sub
strate
s with leaving group
s of differing reactivity, aflat linear free energy relation
ship wa
s produced, indicating that the C4-O4 bond i
s not broken in a rate-determining
step. Taken together, the
se re
sult
s strongly
sugge
st a
stepwi
se mechani
sm. Con
si
stent withthi
s wa
s the mea
surement of a
secondary deuterium kinetic i
sotope effect upon
kcat/
Km of 1.01 ± 0.03 ona 4-{
2H}-
sub
strate, indicating that no
sp
2 character i
s developed at C4 during the rate-limiting
step, therebyruling out a concerted
syn-elimination.