C O M M U N I C A T I O N S
Table 1. Kinetic Parameters of PAM with Various Substratesa
the 3′-methoxy isomer was below detection limits, considering that
the weakly electron-donating 3′-methyl substrate 7 was converted
to its â-isomer nearly 4 times faster and with greater efficiency
than the 4′-methyl isomer 5.
Thien-2-yl-(S)-R-alanine (2) was converted to â-amino acid
product much faster (Vrel ) 14) than the furan-2-yl analogue 10
(Vrel ) 0.02, Table 1). The efficiency of PAM with 2 was second
to that of the 4′-fluoro species 1 and was also isomerized ∼40 times
more efficiently than natural substrate 6. The KM value for 2 (35
µM) compared to that of 10 (410 µM) indicates that 2 likely has
better binding affinity.
Intriguingly, (S)-styrylalanine (11) was also a productive substrate
of PAM with a Vrel ) 0.041 compared to 6 (Table 1), and its KM
was similar to the meta-substituted analogue 7, although the catalytic
efficiency (Vmax/KM , 0.0001 nmol‚h-1‚µM-1) was much lower.
Notably, PAM was unable to convert the saturated styrylalanine
analogue ((S)-2-amino-5-phenylpentanoic acid) to its â-isomer,
suggesting that an extended conjugated allyl π-system next to the
R-carbon, bearing the migrating amino group, is required for
isomerization.
In conclusion, it is evident that the native PAM can accept
arylalanine substrates with various substituents on the phenyl ring.
However, no definitive trend in the catalytic rate emerged in parallel
with strong electron-withdrawing or -donating substituents at steady-
state. In this scenario, substrate release or rotation dynamics during
the reaction may be rate-limiting and thus masks cryptic inductive
effects associated with the MIO mechanism. Single turnover kinetic
analysis of PAM is currently being developed to further dissect
the mechanism.
a Vrel is the ratio of Vmax of a particular substrate and Vmax of
phenylalanine. Catalytic efficiency is defined by Vmax/KM. Refer to Scheme
1 for position of R substituent. Standard deviation in parentheses.
As the properties of the phenylalanine aminomutase mechanism
are better understood, the integration of amino acid isomerase
chemistry into custom asymmetric synthesis becomes practical.
Engineering PAM to broaden its substrate specificity for the
production of novel nonpeptidic â-amino acids could provide
building blocks for constructing second-generation compounds.
respectively, were observed (Table 1), while a Vrel at 0.058 and
catalytic efficiency at 0.0022 nmol‚h-1‚µM-1 for the isomerization
of the 2′-methyl regioisomer 9 was substantially lower. Interestingly,
the KM (50 µM) for 9 is lowest in the methyl series, indicating that
substrate binding is likely not the rate-determining factor of the
methylated species. Thus, the ortho-methyl group (cf. Figure 1)
may restrict conformational dynamics of reaction intermediates that
are less affected by the meta- and para-methyls during reaction
progress.
Acknowledgment. This work was supported by the Michigan
State University College of Arts and Science and by the Michigan
State University Agricultural Experiment Station.
The Vmax of PAM for 4′-tert-butyl-(RS)-R-phenylalanine (12) was
slower (Vrel , 0.001) than that of 6; however, the modest
isomerization of 12 reveals that the PAM active site can accom-
modate a relatively bulky, aliphatic substituent at the para-position
of the substrate (cf. Figure 1). However, the nine -C-C-H bond
extensions of the tert-butyl group likely place 12 (KM ≈ 1000 µM)
near the steric limits of the active site and affect binding compared
to the smaller 4′-methyl homologue 5 (KM ) 160 µM).
Supporting Information Available: Experimental procedures, mass
spectral analysis (Figure 1S), and the enantiopurity of the biosynthetic
products (Figure 2S). This material is available free of charge via the
References
(1) Garcion, E.; Lamprecht, A.; Heurtault, B.; Paillard, A.; Aubert-Pouessel,
A.; Denizot, B.; Menei, P.; Benoit, J.-P. Mol. Cancer Ther. 2006, 5 (7),
1710-1722.
Phenylalanine analogues bearing electron-withdrawing or electron-
donating groups on the aromatic ring were used to investigate the
effects of electron induction on PAM at steady-state. The catalytic
efficiency of PAM catalysis for the 4′-fluoro-R-regioisomer (1) at
2.4 nmol‚h-1‚µM-1 was greatest of all of the substrates tested. The
(2) Belyaev, N. A.; Kolesanova, E. F.; Kelesheva, L. F.; Rotanova, T. V.;
Panchenko, L. F. B. Exp. Biol. Med. 1990, V110 (5), 1483-1485.
(3) Weaver, D. F.; Tan, C. Y. K.; Kim, S. T.; Kong, X.; Wei, L.; Carran, J.
R. Antiepileptogenic agents; WO 2002073208, 20020313, 2002.
(4) Fueloep, F.; Martinek, T. A.; Toth, G. K. Chem. Soc. ReV. 2006, 35 (4),
323-334.
(5) Cardillo, G.; Tomasini, C. Chem. Soc. ReV. 1996, 25 (2), 117-128.
(6) Tan, C. Y. K.; Weaver, D. F. Tetrahedron 2002, 58 (37), 7449-7461.
2′-fluoroisomer4waslessefficientlycatalyzed(0.027nmol‚h-1‚µM-1
)
by ∼60-fold compared to 1, while the 3′-fluoro isomer 8 was even
less efficiently isomerized (0.0057 nmol‚h-1‚µM-1) by ∼400-fold
(Table 1). The turnover of each fluorinated substrate was greater
than that of phenylalanine. In general, an electron-withdrawing
fluoro on the ring of the substrate may increase the acidity of the
âH of the substrate (cf. Figure 1), thus increasing the overall reaction
rate, and therefore supports pathway B in Figure 1. In contrast,
however, substrate 3 with an electron-donating 4′-methoxy group
also demonstrated greater efficiency (0.12 nmol‚h-1‚µM-1) and
superior turnover (Vrel ) 25) compared to that of 6 (Table 1), thus
supporting pathway A in Figure 1. Surprisingly, the turnover of
(7) Walker, K. D.; Klettke, K.; Akiyama, T.; Croteau, R. J. Biol. Chem. 2004,
279 (52), 53947-53954.
(8) Steele, C. L.; Chen, Y.; Dougherty, B. A.; Li, W.; Hofstead, S.; Lam, K.
S.; Xing, Z.; Chiang, S.-J. Arch. Biochem. Biophys. 2005, 438 (1), 1-10.
(9) Calabrese, J. C.; Jordan, D. B.; Boodhoo, A.; Sariaslani, S.; Vannelli, T.
Biochemistry 2004, 43 (36), 11403-11416.
(10) Christenson, S. D.; Wu, W.; Spies, M. A.; Shen, B.; Toney, M. D.
Biochemistry 2003, 42 (43), 12708-12718.
(11) Asano, Y.; Kato, Y.; Levy, C.; Baker, P.; Rice, D. Biocatal. Biotransform.
2004, 22 (2), 133-138.
(12) Ege, M.; Wanner, K. T. Org. Lett. 2004, 6 (20), 3553-3556.
JA071328W
9
J. AM. CHEM. SOC. VOL. 129, NO. 22, 2007 6989