6942
J . Am. Chem. SOC.1983, 105, 6942-6943
Stereochemistry of the Phosphorylation Reaction of a-Chymotrypsin by a Cyclic Phosphate Triester Dinesh 0. Shah, Deborah Kallick, Robert Rowell, Rouhlwai Chen, and David G . Gorenstein* Contributionfrom the Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois 60680. Received January 24, 1983
Abstract: The reaction of a-chymotrypsin with the axial triester 2-(2,4-dinitrophenoxy)-2-oxo-truns-5,6-tetramethylene1,3,2-dioxaphosphorinane( l a ) yields a stable serine ester enzyme complex. The 31PN M R spectrum of the native and denatured triester complex shows a single peak a t ca. -4.7 ppm. 31P NMR of synthetic model compounds 2-(N-~arbobenzoxy-~-serine methyl ester)-2-oxo-trans-5,6-tetramethylene-l,3,2-dioxaphosphorinanes (3a,b) shows two peaks at -5.8 and -4.7 ppm under the same conditions corresponding to the axial epimer (3a) and equatorial epimer (3b), respectively. This suggests that the enzymatic reaction a t phosphorus in compound l a proceeds with 100% inversion of configuration.
The enzymatic stereochemistry of reaction a t phosphorus has been of considerable current interest, and so far in all enzymatic phosphoryl transfer reactions inversion of configuration at phosphorus in single-displacement steps has been Observation of overall retention of configuration in an enzymatic reaction has also been observed. However, this has been interpreted a s evidence for formation of a transient covalent phosphorylated enzyme intermediate, with each phosphoryl transfer step (phosphorylation of t h e enzyme followed by dephosphorylation) proceeding with inversion of configuration a t phosphorus. The double-displacement enzymatic reaction thus gives a product of net retention of configuration. Overall retention stereochemistry could equally well result if each of t h e two steps proceeded with retention of configuration. In none of t h e double-displacement enzymatic reactions has it been proven, however, that the individual phosphoryl transfer steps proceed with retention or inversion of configuration. In this paper we provide t h e first direct proof t h a t a t least t h e first step involving phosphorylation of a n enzyme (although one not involving a normal substrate) does indeed proceed with inversion of configuration. Phosphorylated derivatives of a-chymotrypsin have provided important structural and mechanistic information on this enzyme.I0 Thus, the diisopropylphosphoryl-a-chymotrypsinspecies, in which serine- 195 is covalently phosphorylated, has contributed t o our understanding of t h e important nucleophilic role played by t h e y-oxygen of serine- 195 in the enzymatic mechanism of hydrolysis of acyl substrates.” Indeed, t h e tetrahedral phosphorylated enzyme may be considered a “transition-state analogue” of the tetrahedral carbonyl addition intermedialeltransition state.I2J3
Experimental Section General Procedures. 31PN M R spectra were recorded on a Bruker WP-80 spectrometer at 32.4 MHz (”P) and ‘H N M R on a 60-MHz Varian T-60 spectrometer. Chemical shifts in parts per million for ‘H N M R spectra are referenced to external Me,Si and for ,IP N M R spectra are referenced to 85% H3P04. Mass spectra were taken on an AEI MS-30 spectrometer. Infrared spectra were obtained on a Perkin-Elmer 727 B spectrometer. Melting points were taken on a Thomas-Hoover apparatus and are uncorrected. Chemicals were generally of the highest purity. Baker Analyzed 60-200 mesh silica gel was used for column chromatography after being activated at 130 OC overnight. Pyridine was refluxed over and then distilled from calcium hydride. Diethyl ether was refluxed over and distilled from sodium metal. a-Chymotrypsin (Bovine pancreatic) was purchased from Sigma Chemical Co. as a 3 times crystallized and lyophilized salt-free type I1 powder. Active site titration of the enzyme followed the method of Schonbaum et al.’* and routinely yielded 80-85% active sites. Protease type XIV (Pronase E) was purchased from Sigma Chemical Co. The axial and equatorial epimers of 2-(2,4-dinitrophenoxy)-2-0~0rruns-5,6-tetramethylene-l,3,2-dioxaphosphorinane[2-(2,4-dinitrophenoxy)-l,3-dioxa-2-phospha-truns-decalin-2-one] (la,b) were prepared as described by Gorenstein et aI.I4 0-2 A- ON P
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(1) Eckstein, F.; Goody, R. S . Biochemistry 1976, 15, 1685. (2) Eckstein, F. Acc. Chem. Res. 1979, 12, 204. (3) Richards, J. P.; Frey, P. A. J . Am. Chem. SOC.1978, 100, 7757. (4) Abbott, S. J.; Jones, S. R.; Weinman; S . A,; Knowles, J. R. J . Am. Chem. SOC.1978, 100, 2558. (5) Knowles, J. R., Annu. Reu. Biochem. 1980, 49, 877. (6) Tsai, M. D. Biochemistry 1979, 188 1468. Tsai, M. D. ‘P-31 NMR: Principles and Applications”; Gorenstein, D. G., Ed.; Academic Press: New .. York,‘in press. (7) Gerlt, J. A.; Coderre, J. A. J . Am. Chem. SOC.1980, 103, 4531. Gerlt, J. A. “P-31 NMR: Principles and Applications”; Gorenstein, D. G., Ed.; .. Academic Press: New Yoik, in press. (8) Lowe, G.; Potter, B. V. L., Biochem. J . 1981, 199, 693. (9) Webb, M. R.; Trentham, D. R. J . Biol. Chem. 1980, 255, 8629. (10) Blow, D. M. “The Enzymes”, 3rd ed.; Academic Press: New York, 1971; Vol. 3, pp 185-212 and references therein. Blow, D. M. Acc. Chem. Res. 1976, 9, 145. (11) Jansen, E. F.; Nutting, M-D. F.; Balls, A. K. J . Biol. Chem. 1979, 179, 189. (12) Wolfenden, R. Acc. Chem. Res. 1972, 5, 10. (13) Lienhard, G. E. Annu. Rep. Med. Chem. 1972, 7, 249. (14) Gorenstein, D. G.; Rowell, R.; Findlay, J. J . Am. Chem. SOC.1980, 102, 5077. (15) Rowell, R.; Gorenstein, D. G. J. Am. Chem. SOC.1981, 103, 5894.
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1: R=2,4-DNP 3: R=CH2CH (C02CH3) NHCBZ 2-Oxo-trans -5,6-tetramethylene-1,3,2-dioxaphosphorinane-~-chymotrypsin Complex, 2. The axial phosphorinane epimer, l a (7.5 mg), was added slowly to 100 mg of 3 times recrystallized a-chymotrypsin in 20 mL of 11% acetonitrile, aqueous Tris (0.5 M) buffer, pH 7.8, with stirring at room temperature (some preparations used 10% dioxane as cosolvent instead of the acetonitrile). After overnight stirring, an additional 7.5 mg of l a was added and left for an additional 6 h. The sample was adjusted to pH 3 and centrifuged, and the supernatant was
(16) Gorenstein, D. G. ‘P-31 NMR: Principles and Applications”; Academic Press: New York, in press. (17) Gorenstein, D. G.; Rowell, R. J . Am. Chem. SOC.1980, 102, 6165. (18) Schonbaum, G. R.; Zerner, B.; Bender, M. L. J . Biol. Chem., 1961, 236, 2930.
0002-7863/83/1505-6942$01.50/0 0 1983 American Chemical Society
J . A m . Chem. SOC.,Vol. 105, No. 23, 1983 6943
Phosphorylation of a - Chymotrypsin
I
1
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Figure 1. "PI'HJ N M R of denatured enzyme complex 2 in 8 M urea,
Figure 2. "P('HJ N M R of proteolytically cleaved enzyme complex 2 in
2 0 8 D20, pH 7.0, 32.4 MHz.
20% D 2 0 , 1 mM EDTA, pH 7.0, 32.4 MHz.
dialyzed overnight at 4 "C against 1 L of pH 3 (1 mM HCI) buffer. The sample was lyophilized. Essentially no enzymatic activity (
