lanthanide micelles (13.1–76.8 M21 s21 19
which have Lewis
)
Notes and references
1 L. Pauling, Am. Sci., 1948, 36, 51.
2 C. Walsh, Enzymatic Reaction Mechanisms, Freeman, New York,
1979.
acidities several orders of magnitude higher.20 The specific
activities of 1.6–9.7 nmol min21 mg21 against 1 mM NPPP
(Table 1) are comparable to those of a few phosphatases and
phosphodiesterases at 30 °C (12–355 and ca. 1–38 900
nmol min21 mg21, respectively).15 Conversely, the hydrolyses
of the phosphomonoester p-nitrophenylphosphate and the
phosphotriesters parathion and tris(p-nitrophenyl)phosphate are
beyond the spectrophotometric detection limit, indicating the
presence of specificity toward different phosphoesters.
3 K. Nishida, Y. Ohta, M. Ito, Y. Nagamura, S. Kitahara, K. Fujii and I.
Ishiguro, Biochim. Biophys. Acta., 1996, 1313, 47; C. V. Preuss and
C. K. Svensson, Biochem. Pharmacol., 1996, 51, 1661; L. Luan, T.
Sugiyama, S. Takai, Y. Usami, T. Adachi, Y. Katagiri and K. Hirano,
Biol. Pharmacol. Bull., 1997, 20, 71.
4 N. Sträter and W. N. Lipscomb, Biochemistry, 1995, 34, 9200; B.
Lejczak, P. Kafarski and J. Zygmunt, Biochemistry, 1989, 28, 3549.
5 D. E. Tronrud, H. M. Holden and B. W. Matthews, Eur. J. Biochem.,
1986, 157, 261.
6 E. M. Prager and A. C. Wilson, J. Mol. Evol., 1988, 27, 326; K. Nitta and
S. Sugai, Eur. J. Biochem., 1989, 182, 111; H. A. McKenzie and F. H.
White, Jr., Adv. Protein Chem., 1991, 41, 173; P. K. Qasba and S.
Kumar, Crit. Rev. Biochem. Mol. Biol., 1997, 32, 255.
7 C. S. Bond, P. R. Clements, S. J. Ashby, C. A. Collyer, S. J. Harrop,
J. J. Hopwood and J. M. Guss, Structure, 1997, 5, 277; G. Lukatela, N.
Krauss, K. Theis, T. Selmer, V. Gieselmann, K. von Figura and W.
Saenger, Biochemistry, 1998, 37, 3654.
Although the p-nitrophenol in both BNPP and NPPP is a very
good leaving group, the auto-hydrolytic rates of BNPP and
NPPP are still extremely slow with a rate constant k1 = 1.1 3
10211 s21 for BNPP at pH 7.0 and 25 °C21 and 7.65 3 1028 s21
for NPPP at pH 8.0 and 50 °C (comparable to 1.7 3 1027 s21
at 60 °C18). Tremendous catalytic proficiencies22 are obtained
for M2-sAP toward BNPP and NPPP hydrolyses i.e. (0.94–67)
3 109 and (0.43–2.9) 3 105, respectively (Table 1). Co22sAP
virtually decreases the half-life of BNPP hydrolysis from ca.
2000 years to ca. 1 second! These rate enhancements are
remarkable when it is taken into account that the phospho-
substrates are transition-state analogues of peptides during
hydrolysis1,3–5 In this case their corresponding trigonal bipyr-
amidal transition states requires significantly more stabilization
in support of their hydrolysis. For instance, an association
constant of 108 M21 (approximated from the average Km of
9.3 mM for BNPP hydrolysis) would contribute 11.6 kJ mol21
energy in ground-state stabilization at 298 K, which would
increase the activation energy by the same amount and would
reduce the reaction rate by ca. 1003. In the mean time, a 67 3
109 fold rate enhancement requires a decrease of 61.7 kJ mol21
in activation energy at 298 K.
8 R. Pickersgill, G. Harris, L. Lo Leggio, O. Mayans and J. Jenkins,
Biochem. Soc. Trans., 1998, 26, 190.
9 P. J. O’Brien and D. Herschlag, J. Am. Chem. Soc., 1998, 120,
12 369.
10 H. I. Park and L.-J. Ming, Angew. Chem., Intl. Ed., 1999, 38, 2914.
11 The purification of sAP (ca. 30 kDa) and preparation of its apo form
followed the literature procedures.11a,b,12 The kinetic measurements by
the metal-substituted derivatives were conducted in the presence of
excess amount of the corresponding metal ions to ensure the complete
formation of the derivatives. The background hydrolysis of BNPP by the
excess metal ion is negligible and that of NPPP is considerably small
and has been corrected. (a) A. Spungin and S. Blumberg, Eur J.
Biochem., 1989, 183, 471; (b) D. Ben-Meir, A. Spungin, R. Ashkenazi
and S. Blumberg, Eur J. Biochem., 1993, 212, 107.
Finally, it is interesting that the Mn2+, Ni2+ and Cd2+
derivatives of sAP also exhibit potent hydrolytic power [i.e. (ca.
1–4) 3 109 and (ca. 40–200) 3 103 fold rate enhancements
toward BNPP and NPPP hydrolyses, respectively, Table 1]
suggesting that these metal ions should be included in future
design of chemical models for more extensive structural and
mechanistic studies of metal-centered hydrolysis.23
The results have provided some mechanistic insight. The Km
values for BNPP and NPPP are similar, suggesting that they are
recognized by sAP in a similar fashion. In both substrates, a
hydrophobic p-nitrophenyl/phenyl group (as a hydrophobic
anchor to bind to the active site) and a –PO22– group (as a gem-
diolate transition-state analogue) are essential. On the other
hand, the non-competitive property of phosphate24 may be
attributable to the lack of a hydrophobic anchor; the competitive
inhibitor p-nitrophenylphosphate25 contains both recognition
moieties, yet is not hydrolyzed owing to the lack of an
additional hydrolyzable group; and the two phosphotriesters are
not hydrolyzed owing to the lack of a –PO22– group.
Many synthetic metal complexes have been utilized as
models26 to provide insight into the mechanistic roles of the
metal ion(s) and the nucleophilic water in metallohydrolases.
The specificity and tremendous effectiveness of enzymes offer
a very challenging task for chemical modeling studies to
achieve. We describe here that the transition-state analogues
BNPP and NPPP are indeed substrates for M2-sAP, and are
hydrolyzed with remarkable rate accelerations. Although the
rates of catalysis are not comparable to those of ‘perfect
enzymes’ and much slower than those of the specific substrates
of sAP,11a,b M2-sAP can serve as unique ‘natural model
systems’ (as opposed to ‘synthetic model systems’) for further
studies of phosphoester hydrolysis. The results from these
studies should lead us to a better understanding of dinuclear
hydrolysis in chemical and biological systems.
12 L.-Y. Lin, H. I. Park and L.-J. Ming, J. Biol. Inorg. Chem., 1997, 2,
744.
13 T. Koike and E. Kimura, J. Am. Chem. Soc., 1991, 113, 8935; E.
Kimura, H. Hashimito and T. Koike, J. Am. Chem. Soc., 1996, 118,
10 963.
14 Second-order rate constants in the range of (0.18–2.8) 3 1025 M21 s21
are calculated from corresponding pseudo-first order rate constants at
pH 8.36 and 55 °C14a and (5.4–11.5) 3 1025 M21 s21 at pH 10.9–11.5
and 35 °C14b for several mono- and di-nuclear Zn2+ complexes.
(a) W. H. Hapman and R. Breslow, J. Am. Chem. Soc., 1995, 117, 5462;
(b) A. Bencini, E. Berni, A. Bianchi, V. Fedi, C. Giorgi, P. Paoletti and
B. Valtancoli, Inorg. Chem., 1999, 38, 6323.
15 J. S. Kelly, D. E. Dardinger and L. G. Butler, Biochemistry, 1975, 14,
4983; J. S. Kelly and L. G. Burtler, Biochem. Biophys. Res. Commun.,
1975, 66, 316.
16 H. Shim, S.-B. Hong and F. M. Raushel, J. Biol. Chem., 1998, 273,
17 445.
17 Y. C. Yang, J. A. Baker and J. R. Ward, Chem. Rev., 1992, 92, 1729.
18 NPPP hydrolysis by La3+ is enhanced only by 100-fold at 60 °C which
is much smaller than the catalytic proficiency of sAP (Table 1), whereas
Cu2+, Ni2+ and Zn2+ are ineffective; J. S. Loran, R. A. Naylor and A.
Williams, J. Chem. Soc., Perkin Trans. 2, 1977, 418.
19 R. Moss and K. G. Ragunathan, Langmuir, 1999, 15, 107.
20 J. Burgess, Metal Ions in Solution, Halstead, New York, 1978.
21 B. K. Takasaki and J. Chin, J. Am. Chem. Soc., 1995, 117, 8582.
22 The catalytic proficiency is expressed as kcat/k122a instead of (kcat/Km)/
(k1/55.5),9 which is not appropriate here since H2O is not the
nucleophile in the hydrolysis. (a) A. Radzicka and R. Wolfenden,
Science, 1995, 26, 90.
23 Recent references: F. Hampl, F. Liska, F. Mancin, P. Tecilla and U.
Tonellato, Langmuir, 1999, 15, 405; J. Suh and W. J. Kwon, Bioorg.
Chem., 1998, 26, 103; C. He, V. Gomez, B. Spingler and S. J. Lippard,
Inorg. Chem., 2000, 39, 4188.
24 M. N. Harris and L.-J. Ming, FEBS Lett., 1999, 455, 321.
25 H. I. Park, Ph.D. Dissertation 1999, University of South Florida, FL,
USA.
26 Some recent reviews: E. Kimura and T. Koike, Adv. Inorg. Chem., 1997,
44, 229; E. L. Hegg and J. N. Burstyn, Coord. Chem. Rev., 1998, 173,
133; N. H. Williams, B. Takasaki, M. Wall and J. Chin, Acc. Chem. Res.,
1999, 32, 485; A. Blasko and T. C. Bruice, Acc. Chem. Res., 1999, 32,
475; H. Vahrenkamp, Acc. Chem. Res., 1999, 32, 589.
This research was partially supported by the Petroleum
Research Funds administrated by the American Chemical
Society (ACS-PRF #35313-AC3).
2502
Chem. Commun., 2000, 2501–2502