Tian et al.
785
and earlier views we expressed (12), we suspect that a low-
barrier hydrogen bond stabilizes the transition state here.
within 1.2–1.7% of the densities estimated as the weighted
average of the densities of the pure solvents. To obtain the
activities of water a in mixtures of acetonitrile and water,
w
the data of Vierk (14) and of Maslan and Stoddard (15) at 20
and 30°C were fitted to third-order polynomial functions in
the mole fraction of acetonitrile. Values at other tempera-
tures were interpolated or extrapolated assuming a linear re-
Conclusions
The fact that the proton inventories and solvent isotope ef-
fects generally remain linear over a considerable range of
solvent compositions suggests that the medium plays no role
in controlling the magnitude or character of the largest con-
tribution to the solvent isotope effects and proton invento-
ries (see below for smaller contributions). If there were
actually multiple contributions to the major part of the iso-
tope effect from generalized medium effects, then it would
seem likely that strong variations in the composition of the
medium would lead to non-linearity in the proton invento-
ries as the medium-dependent part of the isotope effect was
changed. The conclusion that the secondary site (at the ques-
tion mark in 1 does not generate an isotope effect or partici-
pate in catalysis for these deacylation reactions therefore
seems justifiable.
lation between ln a and 1/T. The effective pH in mixtures
w
of acetonitrile and water, pH*, was estimated by the method
of Perrin and Dempsey (16) according to which pH* = R – δ,
where R is the apparent pH as read from a pH-meter
equipped with a glass electrode. Values of δ were available
from the work of Douheret (17) for some mixtures of
acetonitrile and water. These values were fitted to a third-
order polynomial in the mole fraction of acetonitrile so that δ
could be obtained for other mixtures by interpolation or ex-
trapolation. The magnitude of δ never exceeded 0.1–0.4. The
effective pH in mixtures of protium and deuterium oxides
was obtained from the customary correction to the glass-
electrode reading (3c).
There are two exceptions to the observation of constant
isotope effect and identical proton inventory in different me-
dia. These exceptions are the shifts from an isotope effect of
Kinetics
Initial rates of enzyme-catalyzed hydrolysis were deter-
mined as slopes of absorbance at 400 nm vs. time. Reaction
solutions of 3 mL volume in a quartz cuvette, lacking only
enzyme, were thermally equilibrated in the cell compartment
of an HP 8542A diode-array spectrophotometer and reaction
was initiated by injection of 10 µL of enzyme stock solution.
After manual mixing, data collection was initiated. Initial
rates in absorbance units were converted to molar units on
the basis of measured extinction coefficients for acetonitrile–
water mixtures. For the range 0–30% (v/v) acetonitrile, the
extinction coefficients were constant within 0.1%, so that
2
.5 to an isotope effect of 3.0 as the acetonitrile content falls
from higher values to 15% for the Trp acyl enzyme and
from an isotope effect of 2.2 to one of 2.5 as the acetonitrile
content falls from higher values to 16% for the Leu acyl en-
zyme. Both changes in the isotope effect are relatively small
(
factors of 1.2 and 1.1, respectively) and may in fact signal
the incursion of medium effects of the order of 10–20% as
the medium approaches pure water. However, the effects are
in the same direction as the single-site effect and would
therefore not conceal any normal isotope effect from a sec-
ondary-site contribution. Thus the absence of a secondary-
site contribution appears a reasonable conclusion.
–
1
–1
the average value of 9830 M cm was used for all cases.
Values of kcat and kcat / K were obtained by weighted least-
m
Although there seem to be no generalized medium effects
at work in the enzymic deacylation reactions examined here,
they certainly exist. Stein et al. (13) and Kresge and co-
workers (7) found such effects, for example, in the acylation
reactions of the serine proteases. Therefore, Kresge’s cri-
tique (5) is in general one that requires attention.
squares fitting of specific initial velocities (initial rate/total
enzyme concentration) to substrate concentrations in a dou-
ble-reciprocal (Lineweaver–Burk) formulation.
References
1
2
. A.J. Kresge. Pure Appl. Chem. 8, 243 (1964).
. C.R. Hopper, R.L. Schowen, K.S. Venkatasubban, and H.
Jayaraman. J. Am. Chem. Soc. 95, 3280 (1973).
Experimental
3
. (a) K.B. Schowen. In Transition states of biochemical pro-
cesses. Edited by R.D. Gandour and R.L. Schowen. Plenum
Press, New York. 1978. Chap. 6. pp. 225–283; (b) K.S.
Venkatasubban and R.L. Schowen. Crit. Rev. Biochem. 17, 1
Materials
Buffer salts and spectranalyzed acetonitrile were obtained
from Fisher Scientific Co. Deuterium oxide (99.9%) was ob-
tained from Aldrich Chemical Co. Substrates were obtained
from Sigma Chemical Co. and stored in a desiccator below
(
1984); (c) K.B. Schowen and R.L. Schowen. Methods
Enzymol. 87C, 551 (1982); (d) D.M. Quinn and L.D. Sutton.
In Enzyme mechanisms from isotope effects. Edited by P.F.
Cook. CRC Press, Boca Raton, Fla. 1991. pp. 73–126.
0
°C. Bovine pancreatic α-chymotrypsin (EC 3.4.21.1) was
obtained from Sigma Chemical Co. as a salt-free, lyophilized,
three-times recrystallized powder with an activity of 42
BTEE units/mg of solid and was stored in a desiccator
below 0°C. Distilled water was deionized on an ion-
exchange column before use.
4
. E. Pollock, J.L. Hogg, and R.L. Schowen. J. Am. Chem. Soc.
9
5, 968 (1973).
5
6
. A.J. Kresge. J. Am. Chem. Soc. 95, 3065 (1973).
. Y. Chiang, A.J. Kresge, T.K. Chang, M.F. Powell, and J. Wells.
J. Chem. Soc. Chem. Commun. 1587 (1995).
. T.K. Chang, Y. Chiang, H.-X. Guo, A.J. Kresge, L. Mathew,
M.F. Powell, and J.A. Wells. J. Am. Chem. Soc. 118, 8802
(1996).
Solutions
7
Buffers were prepared in mixtures of protium oxide, deu-
terium oxide, and acetonitrile with constant weights of
buffer acid and buffer base. Densities of water–acetonitrile
mixtures were determined pycnometrically and found to be
8. (a) T. Ke and A.M. Klibanov. J. Am. Chem. Soc. 120, 4259
(1998); (b) K. Kawashiro, W.J. Moree, P. Sears, K. Witte, and
©
1999 NRC Canada