Abstract
The benzoxazinones 2-ethoxy-4H-3,l-benzoxazin-4-one (la) and 2-(trifluoromethyl)-4/f-3,1 -benzoxazin-4-one (Id) inactivate chymotrypsin. The inactivation
is stoichiometric and proceeds with rate constants of 7 X 105
M"1 min'1 and >4 X 106 M'1 min'1, respectively. The inactivated enzyme recovers catalytic activity slowly, k =
2.3 X
10'3 min"1 and 3.7 X 10'2 min"1 (pH 7.1). When the enzyme
regains catalytic activity, 2-[JV-(ethoxycarbonyl)amino]benzoic
acid is released from enzyme inactivated with la and N-
(trifluoroacetyl)anthranilic acid from enzyme inactivated with
Id. The mechanism of inactivation involves attack of the active
site serine on the C-4 carbonyl of the inactivator which leads
to ring opening and formation of an ortho-substituted benzoylchymotrypsin, which hydrolyzes slowly due to electron
releasing ability of the substituents. The rate of hydrolysis
of the benzoylchymotrypsin from la or Id is in close agreement
with those predicted from the Hammett parameters ( , p) for
hydrolysis of their para-substituted analogues [Caplow, M.,
& Jencks, W. P. (1962) Biochemistry 1, 883-893]. The
inactivation of chymotrypsin by 2-methyl-4/f-3,l-benzoxazin-4-one (lb) is an equilibrium process (fcinact = 1 X 104
M"1 min"1 and KVi =
2 X 106 M"1). Formation of a benzoylchymotrypsin is demonstrated by spectral changes and
methanol trapping. The benzoylchymotrypsin can also decay
by direct hydrolysis to iV-acetylanthranilic acid. Chymotrypsin
is also inactivated by 2-amino-4Ff-3,1 -benzoxazin-4-one (lc).
The inactivation is rapid (k =
8.7 X 104 M"1 min"1), but
reactivation is also relatively fast (k =
0.14 min'1). Upon
reactivation benzoyleneurea is released. Reaction of lc with
chymotrypsin leads to the formation of (o-ureidobenzoyl)-
chymotrypsin, which does not undergo hydrolysis but decomposes through intramolecular attack of the ureido NH2 upon
the carbonyl group of the benzoyl ester. Compound la also
inactivates trypsin and elastase. Compound lb was a poor
inactivator of trypsin and does not inhibit elastase. Both
trypsin and elastase catalyze the conversion of lc to benzoyleneurea.