J. Phys. Chem. 1984,88,6021-6024 TABLE II: Previous Results for the HOz Low-Pressure Limit at 298 K
ref 15 3
technique" DF/MS DF/LMR
6, 7 4
FP/IR MM/UV
1 5 8
FP/UV FP/UV DF/LMR DF/MS-UV
this work
+ HOZReaction in the
pressure, torr 0.6 2.0 3.0 4.0 7-20 3.0 5.3 10 b
k l = 2.3
1O1*kl,om3 molecule-' s-' 0.29 & 0.12 0.43 f 0.18 0.55 f 0.14 1.6 f 0.1 1.1 1.5 1.8 1.6 f 0.2 1.4 & 0.2 1.5 f 0.3 1.5 & 0.4
1-7 1
+
exp((600 f 130)/7') 8.4 X 10-34[Ar] exp((1100 f 300)/T)
Our earlier study' indicating that the observed rate constant extrapolates to a zero-pressure limiting value of (1.6 f 0.2) X cm3 molecule-' s-' agrees with the low-pressure results of Thrush and T~nda11,~~' Simonaitis and H e i ~ k l e n Takacs ,~ and Howard: and this work. The results of Thrush and Wilkinson3 which extrapolate to a zero bimolecular intercept and suggest a large termolecular component appear to conflict with the later flash photolysis work of Thrush and Tynda11.6*7The significant pressure dependence observed by Cox and Burrows4 between 3 and 10 torr also disagrees with the recent group of s t ~ d i e s ' - ~ ~ ~ - ~ which report a much smaller termolecular component. In the only other DF/MS study of this reaction, Foner and 3X cm3 molecule-' s-l in the Hudson's obtained kl low-pressure regime using a low-power electrical discharge in H202 as the H 0 2 source. This value must be considered somewhat uncertain due to a number of experimental problems including heterogeneous reactions and possible secondary chemistry involving the OH radical. In addition, no details were provided concerning the H 0 2 calibration method.
-3
b
X
6021
-
"DF, discharge flow; FP, flash photolysis; MM, molecular modulation; MS, mass spectrometry; UV, ultraviolet absorption; LMR, laser magnetic resonance; IR, infrared absorption. Experiments were carried out at pressures up to 1 atm. Quoted zero-pressure limiting rate constant is obtained from extrapolation of these data. spectrometer ionizer and the additional steps involved in the calibration. It was therefore concluded that UV absorption was the more reliable of the two calibration methods, a t least when using mass spectrometric detection of HOZ. Previous work on the low-pressure room-temperature behavior of the H02 HOz reaction is summarized in Table 11. We have recently foundZthat over the pressure range 50-700 torr (Ar) and temperature range 240-417 K the rate constant fits the expresion
Acknowledgment. We thank J. J. Margitan for several useful discussions and C. J. Howard for communicating his results prior to publication. The research described in this paper was carried out by the Jet Propulsion Laboratory, California Institute of Technology, under contract with the National Aeronautics and Space Administration.
+
Registry No. H 0 2 , 3170-83-0.
Pressure Dependence of the a-Chymotrypsin-Catalyzed Hydrolysis of Amide and Anilldes. Evidence for the Single-Proton-Transfer Mechanism Syoichi Makimoto, Keizo Suzuki, and Yoshihiro Taniguchi* Department of Chemistry, Faculty of Science and Engineering, Ritsumeikan University, Kita- ku, Kyoto, 603, Japan (Received: June 15, 1983)
The rates of hydrolysis of N-acetyl-L-tryptophanamide(ATA), N-acetyl-L-tryptophan p-chloroanilide (ATpCA), and succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide (SApNA), catalyzed by a-chymotrypsin (a-CHT), were measured at pressures up to 2 kbar at 25 OC in Tris buffer solutions. Three substrates follow the kinetics of the Michaelis-Menten type. From the pressure dependence of the Michaelis constants KM,the volume changes AVKMwere -3 2 cm3/mol for ATA, 12 f 2 cm3/mol for ATpCA, and 5 2 cm3/mol for SApNA. From the pressure dependence of the rate constant k,, of product formation, the activation volumes AP,,, were -3 2 cm3/mol for ATA, 10 f 2 cm3/mol for ATpCA, and 15 f 2 cm3/mol for SApNA. From the volume changes it is concluded that the reaction mechanism of a-CHT follows single-proton transfer.
*
*
*
introduction A number of investigations' of the reaction mechanism of a-chymotrypsin have led to the conclusion that Ser-195, His-57, and Asp- 102 in the active site contribute to the catalytic reaction. X-ray diffraction studies have suggested a "charge relay systemn2 which forms the hydrogen-bonding (H-bonding) bridges among the carbonyl oxygen of Asp-102, the nitrogen atoms of the imidazole ring of His-57, and the hydrogen atom of Ser-195. The system permits the transfer of negative charge from Asp- 102 to Ser-195 which then acts as a powerful nucleophile in acylation.
On the other hand, kinetic investigations have indicated that there cannot be a normal H bond between the serine and the histidine residues and that simultaneously no H bond between the carboxylate ion and the N H group of the imidazole ring is f ~ r m e d . ~ This H-bonding system (single-proton transfer) results in stabilization3 of the imidazole against a possible release of its proton. Additional kinetic studiese6 of the pH dependence of the hydrolysis of amides have implied that there is an additional intermediate step between the Michaelis complex and the acyl enzyme. In spite of these detailed studies of the mechanism of action of a-CHT, the question still exists as to whether the mechanism is a sin-
(1) Laidlar, K. J.; Bunting, P. S. "The Chemical Kinetics of Enzyme Action", 2nd ed.;Oxford University Press: London, 1973. (2) Blow, D. M.; Birktoft, J. J.; Hartley, B. S . Nature (London)1969,221, 337.
(3) Polger, L.; Bender, M. L. Proc. Narl. Acad. Sci. U.S.A.1969,64, 1335. (4) Caplow, M. J.,Am. Chem. SOC.1969, 91, 3639. ( 5 ) Fersht, A. R.; Requena, Y . J . Am. Chem. SOC.1971, 93, 7079. (6) Fersht, A. R. J. Am. Chem. SOC.1972, 94, 293.
0022-3654/84/2088-6021$01.50/00 1984 American Chemical Society
6022 The Journal of Physical Chemistry, Vol. 88, No. 24, 1984
Makimoto et al.
TABLE I: Kinetic Parameters of the Hydrolysis of Amide and Anilides Catalyzed by a-CHT at Various Pressures and 25 OC 1O3K~/M 102k,,/(M-' s-I) Plkbar ATA" ATpCAb SApNAc ATA' 0.001 0.5 1.0 1.5 2.0
14.7 16.1 16.9 17.3 18.5
1.37 1.12 0.881 0.651
13.3 14.2 10.2 10.1 8.64
9.30 10.2 10.9 11.8 13.4
ATpCAb SApNAc 1.49 1.19 0.990 0.869
3.74 2.55 1.76 1.51 1.11
'pH 7.8, 0.05 M Tris. b p H 7.8, 0.05 M Tris, 10% (v/v) Me2S0. c p H 7.8, 0.1 M Tris.
gle-proton-transfer or two-proton-transfer (charge relay system) mechanism. In order to test these hypotheses, Pollock et al.' reported that the results of isotope effects on the deacylation of acyl-a-CHT indicate a single-proton transfer in the transition state. However, Hunkapiller et ale8suggested the simultaneous transfer of two protons among Asp-102, His-57, and Ser-195 from studies of isotope effects on the hydrolysis of a specific peptide of anilide. The number of proton transfers in the catalytic reaction is related to the number of H bonds formed among Ser-195, His-57, and Asp-102 at the rate determining the transition state. It is well-known that an H-bonding formation accompanies a volume decrease. The volume change for the formation of the H bond ~ ~ ' ~is no of OH- -0type is measured to be -5 ~ m ~ / m o l .There report of a volume change accompanying the formation of NH- e 0 and N. -HO types as with the H bonds between Ser and His or between His and Asp, so that the volume decrease for such Hbonding formation can be calculated from Hamman's method.'' The results are estimated to be -4 to -5 cm3/mol from the decrease of the distance for the H-bonding formation based on X-ray data2 and van der Waals radii'* of atoms related to the H bonding. The change in the water structure for the H-bonding formation is unexpected, as it is clarified that His-57 and Asp-102 are located deep in a hydrophobic pocket of a - C H T from X-ray analysis.' Accordingly, the volume change given by the pressure dependence of the rate process is useful to understand the reaction mechanism of a-CHT. In the present work, the hydrolysis rates of specific substrates, the amide of ATA and the anilides of ATpCA and SApNA, by a-CHT were measured up to 2 kbar at 25 OC. The reaction mechanism of the proton transfer is discussed in terms of volume changes between the substrate binding complexes and the tetrahedral intermediates, and the activation volumes of the acylation. These data are compared with a study of the isotope effects.
-
0
1.0
2.0
P / kbar
Figure 1. Pressure dependence of log KM at 25
OC.
-
Experimental Section Materials. a-Chymotrypsin (a-CHT) (3X crystallized, Sigma Chemical Co.), N-acetyl-L-tryptophanamide(Sigma Chemical Co.), and succinyl-L-alanyl-L-alanyl-L-alanine p-nitroanilide (Nakarai Chemical Co.) were used without further purification. N-Acetyl-L-tryptophan p-chloroanilides was synthesized by the method of Caplow4 and recrystallized 3 times from benzene-ethyl acetate, mp 161-162 OC ( l k 4 mp 162-163 "C). Apparatus and Procedure. The measurement of the hydrolysis reaction up to 2 kbar is described in the previous report.13 The hydrolysis was monitored by the change in the optical density at 306 nm for ATA, 305 nm for ATpCA, and 400 nm for SApNA by means of a Hitachi 340 spectrophotometer under high pressure. (7) Pollock, E.; Hogg, J. L.; Schowen, R. L. J . Am. Chem. SOC.1973,95, 968. (8) Hunkapillar, M. W.; Forgac, M. D.; Richards, J. H. Biochemistry 1976, 15, 5581. (9) Fishrnan, E.; Drickarmer, H.G. J. Chem. Phys. 1956, 24, 548. (10) Taniguchi, Y.; Suzuki, K . J . Phys. Chem. 1974, 78, 759. (1 1) Hamman, S. D. "High Pressure Physics and Chemistry"; Bradley, R. S., Ed.; Academic Press: New York,1963; Vol. 2, Chapter 7. (12) Bondi, A. J . Phys. Chem. 1964, 68, 441. (13) Taniguchi, Y.; Makimoto, S.; Suzuki, K.J . Phys. Chem. 1981, 85, 2218.
0.8
1
0
1 .o
2 ,o
P I kbar Figure 2. Pressure dependence of log k,,, at 25 O C .
The hydrolysis was performed in Tris-HC1 buffer (pH 7.8), 0.05 M for ATA, 0.05 M containing 10% (v/v) MezSO for ATpCA, 0.1 M for SApNA at 25 OC.
Results At each level of pressure, the rates of hydrolysis for the enzyme reaction followed the Michaelis-Menten type kinetics as shown in eq 1 E + S ~KME S % E + P
(1)
where E, S,ES, and P denote enzymes, substrate, the enzymesubstrate complexes, and products, respectively. In the case of [E] > 1,4and eq 3 and 4 change into eq 6 and 7 KM = K,/K
(6)
kcat = k2
(7)
and for amide substrate, K
