COMMUNICATIONS
A 1010 Rate Enhancement of Phosphodiester
Hydrolysis by a Dinuclear AminopeptidaseÐ
Transition-State Analogues as Substrates?**
enabled broad-range optical access for the first time in the near- and mid-
IR region even in a scH2O atmosphere. Aqueous solutions of pinacol
(0.40m) and cyclohexanone oxime (0.15m) were introduced into the system
with flow rates in the range of 0.07 to 2.0 mLmin 1 (the reaction time varied
from 4 to 215 s) using a high-pressure liquid pump, and pressure control was
achieved by a back-pressure regulator. The temperature was then raised to
Hyun Ik Park and Li-June Ming*
1
the desired value. Once the system had stabilized, 50 spectra (4 cm
resolution) were summed in one destination file. Each file was normalized
against the spectrum of pure H2O under the same conditions. Furthermore,
all of the products were identified qualitatively by H NMR spectroscopy
The studies of metal complex based chemical models for
hydrolytic metalloenzymes[1±3] have provided insight into the
mechanistic roles of the metal ion(s) at the active-site and the
coordinated nucleophilic water molecule in those enzymes.[4]
However, these model complexes lack specific recognition
and catalysis toward peptide substrates over phosphoester
substrates, or vice versa, as a result of the absence of a well-
defined active site. Conversely, enzymes have evolved to
recognize one particular type of substrate and are able to
stabilize the corresponding transition state,[5] such as the
tetrahedral transition state in peptide hydrolysis which is very
different from the trigonal bipyramidal transition state in
phosphoester hydrolysis.[4] As a result each class of hydrolytic
metalloenzymes can perform only one type of hydrolysis
despite the presence of a highly activated (>107 in terms of
pKa) coordinated water molecule that is ready for nucleo-
philic attack on the substrate. Moreover, anionic tetrahedral
phosphoesters,[6] phosphonates,[7] phosphoamidates,[8] and
semiacetals,[9] resemble the transition-state gem-diolate of
peptides, esters, and amides formed during hydrolysis and can
serve as potential inhibitors for the corresponding enzymes.
Thus, it seems unlikely that an enzyme with a single active site
can hydrolyze both phosphoester and peptide substrates
because the specific recognitions and hydrolytic pathways
for these two types of substrates are quite different.
Herein we report a unique example of an ªalternativeº
enzyme catalysis in which a dizinc aminopeptidase from
Streptomyces griseus (sAP) exhibits remarkable hydrolytic
activities toward both peptide and phosphodiester substrates.
Thus, this enzyme can serve as an alternative dinuclear model
system to provide further insight into the catalytic mechanism
of metal-centered dinuclear hydrolysis.
The activity profiles obtained during the purification of the
sAP[10, 11] and its thermo-deactivation at 698C show that the
activity toward the hydrolysis of the phosphodiesterase
substrate bis-p-nitrophenylphosphate (BNPP)[12] is always
parallel to the activities toward the aminopeptidase substrates
Leu-p-nitroanilide (Leu-pNA) and Lys-pNA (see data in
ref. [13] and Table 1). This observation suggests that these two
different types of hydrolytic reactions are possibly carried out
by a single enzyme, and the decrease in both activities is
attributable to thermo-denaturation of the sAP. Both activ-
ities are competitively inhibited by the dizinc aminopeptidase
inhibitors bestatin,[14] 1-aminobutylphosphonate,[7] and Leu-
1
and GC-MS.
The frequency precision at the band center of the Raman OH symmetric
stretch (n1) in the spectrum depends on changes in the optical pixel width of
the charge-coupled device, and the resulting frequency error of the n1 mode
1 [8]
is within 2.0 cm
.
Received: March 22, 1999 [Z13199IE]
German version: Angew. Chem. 1999, 111, 3087 ± 3091
Keywords: rearrangements ´ solvent effects ´ supercritical
fluids ´ vibrational spectroscopy
[1] a) NBC/NRC Steam Tables (Eds.: L. Haar, J. S. Gallagher, G. S. Kell),
Hemisphere, New York, 1984; b) High-Temperature Aqueous Solu-
tions: Thermodynamic Properties (Eds.: P. R. J. Fernandez, H. R.
Corti, M. L. Japer), CRC, Boca Raton, FL, 1992; c) R. W. Shaw, T. B.
Brill, A. A. Clifford, C. A. Eckert, E. U. Franck, Chem. Eng. News
1991, 69, 2639; d) W. L. Marshall, E. U. Franck, J. Phys. Chem. Ref.
Data 1981, 10, 295.
[2] a) K. Hatakeda, Y. Ikushima, S. Ito, O. Sato, N. Saito, Chem. Lett. 1997,
245; b) B. M. Kabyemela, T. Adschiri, R. Malaluan, K. Arai, Ind. Eng.
Chem. Res. 1997, 36, 1552.
[3] a) M. Siskin, A. R. Katritzky, Science 1991, 254, 231; b) D. M. Masten,
B. R. Foy, D. M. Harradine, R. B. Dyer, J. Phys. Chem. 1993, 97, 8557;
c) A. R. Katritzky, S. M. Allin, Acc. Chem. Res. 1996, 29, 399; d) J. W.
Schoppelrei, M. L. Kieke, T. B. Brill, J. Phys. Chem. 1996, 100, 7463;
e) M. B. Korzenski, J. W. Kolis, Tetrahedron Lett. 1997, 38, 5611; f) J.
An, L. Bagnell, T. Cablewski, C. R. Strauss, R. W. Trainor, J. Org.
Chem. 1997, 62, 2505; g) O. Sato, Y. Ikushima, T. Yokoyama, J. Org.
Chem. 1998, 63, 9100; h) P. E. Savage, Chem. Rev. 1999, 99, 603; i) K.
Chardler, F. Deng, A. K. Dillow, C. L. Liotta, C. A. Eckert, Ind. Eng.
Chem. Res. 1997, 36, 5175.
[4] a) E. U. Franck, K. Roth, Discuss. Faraday Soc. 1967, 43, 108; b) W.
Kohl, H. A. Lindner, E. U. Franck, Ber. Bunsenges. Phys. Chem. 1991,
95, 1586; c) H. Ohtaki, T. Radnai, T. Yamaguchi, Chem. Soc. Rev. 1997,
41; d) Y. Ikushima, K. Hatakeda, N. Saito, M. Arai, J. Chem. Phys.
1998, 108, 5855.
[5] a) M. C. Burguet, A. Aucejo, A. Corma, Can. J. Chem. Eng. 1987, 65,
944; b) S. Sato, K. Urabe, Y. Izumi, J. Catal. 1986, 102, 99.
[6] a) C. A. Bunton, T. Hadwick, D. R. Liewellyn, Y. Pocker, J. Chem.
Soc. 1958, 403; b) J. F. Duncan, K. R. Lynn, J. Chem. Soc. 1956, 3512;
c) A. Hill, E. W. Flosdorf, Org. Synth. Coll. Vol. 1941, 1, 462; d) T.
Moriyoshi, K. Tamura, Rev. Phys. Chem. Jpn. 1970, 40, 48; e) J.
Boeseken, W. R. van Tonninger, Recl. Trav. Chim. Pays-Bas 1920, 39,
187; f) B. Kuhlman, E. M. Arnett, M. Siskin, J. Org. Chem. 1994, 59,
3098.
[7] D. B. Mitton, P. A. Marrone, R. M. Latanision, J. Electrochem. Soc.
1996, 143, L59, and references therein.
[8] Y. Ikushima, N. Saito, M. Arai, J. Phys. Chem. B 1998, 102, 3029; Y.
Ikushima, K. Hatakeda, N. Saito, M. Arai, Bull. Chem. Soc. Jpn. 1998,
71, 1763.
[9] a) M. M. Hoffmann, S. Conradi, J. Am. Chem. Soc. 1997, 119, 3811;
b) N. Matsubayashi, C. Wakui, M. Nakahara, Phys. Rev. Lett. 1997, 78,
2573.
[10] E. T. Ryan, T. Xiang, K. P. Johnston, M. A. Fox, J. Phys. Chem. 1996,
100, 9395.
[11] a) Y. Ikushima, N. Saito, M. Arai, J. Phys. Chem. 1992, 96, 2293; b) J. B.
Ellington, J. F. Brennecke, J. Chem. Soc. Chem. Commun. 1993, 1094.
[12] C. F. R. Allen, A. Bell, Org. Synth. Coll. Vol. 1955, 3, 312.
[13] A. A. Clifford, K. Pople, W. J. Gaskill, K. D. Bartle, C. M. Rayne,
Chem. Commun. 1997, 595.
[*] Prof. L.-J. Ming, H. I. Park
Department of Chemistry and
Institute for Biomolecular Science
University of South Florida
Tampa, FL 33620-5250 (USA)
Fax: (1)813-974-1733
[**] This work was partially supported by a PYF award (1996) from the
University of South Florida.
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Angew. Chem. Int. Ed. 1999, 38, No. 19