Catalytic enantioselective reductive amination in a host-guest system based on a protein cavity [2]

Full text of DOI:10.1021/ja972771h

1072
J. Am. Chem. Soc. 1998, 120, 1072-1073
cysteine residues. Thus, new cysteines can be easily introduced
into the cavity, and hence, a number of cysteine mutants of IFABP
have been prepared.7 The mutant V60C was particularly interest-
ing to us because it allowed the pyridoxamine moiety to be
introduced deeper in the cavity than was possible with ALBP.8
This region of the protein is rich in aromatic residues including
one tryptophan, three tyrosines, and one phenylalanine as well
as two arginines.9 Modeling experiments suggested that these
residues might form specific contacts with keto acid or amino
acid substrates.
IFABP-PX60 (1, a construct incorporating a pyridoxamine
moiety at poition 60 of IFABP-V60C) was prepared in a similar
manner as previously described for ALBP-PX.5a Following
characterization of the conjugate, the rate of reductive amination
reactions using IFABP-PX60 (50 µM) and R-keto glutarate (3,
50 mM) was studied under single turnover conditions at pH 7.5
and 37 °C; this transformation is shown below.10 Initial results
Catalytic Enantioselective Reductive Amination in a
Host-Guest System Based on a Protein Cavity
Hao Kuang and Mark D. Distefano*
Department of Chemistry, UniVersity of Minnesota
Minneapolis, Minnesota 55455
ReceiVed August 8, 1997
Enzymes are highly efficient catalysts. As a result, many
strategies have been employed to design systems that possess
similar properties.1 Proteins are attractive scaffolds for the design
of new catalysts because their size allows the formation of a large
number of interactions between substrate and catalyst.2 Addition-
ally, recombinant DNA methods allow protein-based catalysts to
be modified in a facile manner either by site-directed mutagenesis
or by selection approaches.3 Fatty acid binding proteins are a
structurally unique family of proteins composed of two orthogonal
â-sheets and a R-helical region.4 These secondary structural
elements fold into a tertiary structure that forms a 600 Å3 cavity
which completely sequesters the fatty acids bound within. In
earlier work, we described the preparation of ALBP-PX, a
construct based on adipocyte lipid binding protein that contained
a pyridoxamine cofactor covalently attached to a cysteine residue
within the cavity.5 This conjugate reductively aminated several
R-keto acids to R-amino acids under single turnover conditions
with excellent enantioselectivities in some cases. However, these
reactions proceeded at rates comparable to those utilizing free
pyridoxamine indicating that the protein scaffold was involved
only in controlling reaction selectivity. To improve this system,
we decided to attach the pyridoxamine moiety to different
positions within the cavity in order to juxtapose the cofactor with
arrays of functional groups present elsewhere within the cavity.
Here, we describe the properties of IFABP-PX60, a catalyst based
on a mutant form of intestinal fatty acid binding protein (IFABP),
a protein structurally related to ALBP.6 This new conjugate
reductively aminates R-keto glutarate to glutamic acid with a
catalytic efficiency at least 200-fold greater than that of free
pyridoxamine. The reaction is catalytic and enantioselective; as
many as 50 turnovers with an enantiomeric purity of 95% ee
(enantiomeric excess) have been obtained.
after 24 h of reaction showed the formation of 1 equiv of glutamic
acid (4) indicating that the reaction was complete; this was quite
different from results obtained in similar experiments performed
with ALBP-PX in which only 46% conversion was observed after
24 h. Clearly, the new conjugate reacts at a more rapid rate.11
The kinetics of this reaction were next analyzed for both the
IFABP-PX60 protein as well as for free pyridoxamine (2). The
results of these experiments are shown in Figure 1. Analysis of
these data using a first-order kinetic model gave a kobsd of 0.18
h-1 for the protein and 0.0029 h-1 for pyridoxamine indicating a
62-fold increase in rate for IFABP-PX60 relative to the free
coenzyme under the same conditions. It should be noted that,
under these single turnover conditions, IFABP-PX60 produced a
68% ee of L-glutamic acid.
In view of the rapid rate obtained under single turnover
conditions, we next examined the ability of the conjugate to
perform this reaction under catalytic conditions in which the
pyridoxamine cofactor is regenerated by the addition of a second
amino acid that serves as an amine source as shown below. The
In our earlier work with ALBP-PX, Cys117 was used as the
site of pyridoxamine attachment. This residue is located near
the C-terminus of the protein and is proximal to the region where
the carboxylate group of the fatty acid ligand binds. To explore
other regions of the protein cavity, we focused on a different fatty
acid binding protein, IFABP, that, unlike ALBP, contains no
(1) Schultz, P. G. Science 1988, 240, 426.
(2) (a) Kaiser, E. T.; Lawrence, D. S. Science 1984, 226, 505. (b) Levine,
H. L.; Nakagawa, Y.; Kaiser, E. T. Biochem. Biophys. Res. Commun. 1977,
76, 64. (c) Corey, D. R.; Schultz, P. G. Science 1987, 238, 1401. (d) Polgar,
L.; Bender, M. L. J. Am. Chem. Soc. 1966, 88, 3153. (e) Neet, K. E.; Koshland,
D. E. Proc. Natl. Acad. Sci. U.S.A. 1966, 56, 1606. (f) Wu, Z. P.; Hilvert, D.
J. Am. Chem. Soc. 1989, 111, 4513. (g) Planas, A.; Kirsch, J. F. Biochemistry
1991, 30, 8268. (h) Wuttke, D. S.; Gray, H. B.; Fisher, S. L.; Imperiali, B. J.
Am. Chem. Soc. 1993, 115, 8455. (i) Imperiali, B.; Roy, R. S. J. Am. Chem.
Soc. 1994, 116, 12083. (j) Suckling, C. J.; Zhu, L. M. Bioorg. Med. Chem.
Lett. 1993, 3, 531. (k) Germanas, J. P.; Kaiser, E. T. Biopolymers 1990, 29,
39. (l) Bell, I. M.; Hilvert, D. New Biocatalysts Via Chemical Modification;
Bell, I. M., Hilvert, D., Eds.; John Wiley & Sons Ltd.: Chichester, 1994; p
73.
(7) Jiang, N.; Frieden, C. Biochemistry 1993, 32, 11015. (b) Frieden, C.;
Jiang, N.; Cistola, D. P. Biochemistry 1995, 34, 2724.
(8) Kuang, H.; Davies, R. R.; Distefano, M. D. Bioorg. Med. Chem. Lett.
1997, 7, 2055.
(9) Sacchettini, J. C.; Gordon, J. I.; Banaszak, L. J. J. Mol. Biol. 1989,
208, 327-339.
(10) For HPLC analysis methods, see: (a) Nimura, N.; Kinoshita, T. J.
Chromatogr. 1986, 352, 169. (b) Buck, R. H.; Krummen, K. J. Chromatogr.
1987, 387, 255. (c) Koh, J. T.; Breslow, R. J. Am. Chem. Soc. 1994, 116,
11234.
(11) Incubation of IFABP-V60C under similar conditions produced no
glutamate product. Addition of 1.0 mM EDTA has no effect on the rate of
reaction with IFABP-PX60 suggesting that rate enhancement via metal ion
catalysis is not occurring. Addition of equimolar Cu(II), Ni(II), or Zn(II)
actually reduces the rate and ee (although these ions accelerate reactions
containing free pyridoxamine).
(3) (a) Knowles, J. R. Science 1987, 236, 1252-1256. (b) Devlin, J. J.;
Panganiban, L. C.; Devlin, P. E. Science 1990, 249, 404-406.
(4) Banaszak, L.; Winter, N.; Xu, Z.; Bernlohr, D. A.; Cowan, S.; Jones,
T. A. AdV. Protein Chem. 1994, 45, 89.
(5) (a) Kuang, H.; Brown, M. L.; Davies, R. R.; Young, E. C.; Distefano,
M. D. J. Am. Chem. Soc. 1996, 118, 10702. (b) Davies, R. R.; Distefano, M.
D. J. Am. Chem. Soc. 1997, 119, 11643.
(6) Lowe, J. B.; Sacchettini, J. C.; Laposata, M.; McQuillan, J. J.; Gordon,
J. I. J. Biol. Chem. 1987, 262, 5931.
S0002-7863(97)02771-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/22/1998

Communications to the Editor
J. Am. Chem. Soc., Vol. 120, No. 5, 1998 1073
Figure 2. Double reciprocal analysis of the initial rates of glutamate
production promoted by IFABP-PX60 and pyridoxamine under catalytic
conditions. Reactions were performed using 50 µM IFABP-PX60 or
pyridoxamine, 5.0 mM tyrosine, and variable amounts of R-keto glutarate
in 200 mM HEPES, pH 7.7, at 37 °C. At selected intervals, samples
were withdrawn and the amount of glutamate was determined by
derivatization and HPLC analysis. Panel A: Reciprocal of rate versus
reciprocal of [R-keto glutarate] for pyridoxamine-catalyzed reaction. Panel
B: Reciprocal of rate versus reciprocal of [R-keto glutarate] for IFABP-
PX60-catalyzed reaction.
Figure 1. Progress curves for glutamate production promoted by IFABP-
PX60 and pyridoxamine under single turnover conditions. Reactions were
performed using 50 µM IFABP-PX60 or pyridoxamine and 50 mM R-keto
glutarate, in 20 mM HEPES, pH 7.5, at 37 °C. At selected intervals,
samples were withdrawn and the amount of glutamate was determined
by derivatization and HPLC analysis. Glutamate production from IFABP-
PX60 (b) and pyridoxamine (O). Also indicated are the production of
L-glutamate (4) and D-glutamate (2) by IFABP-PX60. Glutamate
produced by pyridoxamine is racemic. Curves shown were fit to the
equation [Glu]t ) [Glu]∞{1 - exp(-kobsdt)}.
Table 1. Production of Glutamate in 24 h Catalyzed by
IFABP-PX60 in the Presence of Different Amino Acids
turnovers and an ee of 95% were obtained. Clearly, this protein-
based catalyst is a stable material.
Given the significant increase in rate observed with IFABP-
PX60 compared with free PX, we were interested in clarifying
the origin of this effect. To accomplish this, the rates of
transamination using R-keto glutarate and Tyr were examined at
a range of keto acid concentrations and analyzed using a
Michaelis-Menten kinetic model. The double reciprocal plots
of these data are shown in Figure 2. Analysis of these graphs
gave values for KM and kcat of 150 mM and 0.056 h-1 for PX and
2.9 mM and 0.22 h-1 for IFABP-PX. Comparison of the kcat
values for the protein system versus the free cofactor reveals a
modest 3.9-fold increase resulting from performing the reaction
in the protein cavity. More interestingly, a similar comparison
of the KM values shows a 52-fold decrease suggesting that the
protein conjugate binds R-keto glutarate with much greater affinity
than free pyridoxamine.12 Thus, it appears that the accelerated
catalysis observed with IFABP-PX60 occurs primarily due to an
increase in substrate binding together with a smaller effect on
the maximal rate. Evaluation of the improvement in catalytic
efficiency using kcat/KM as a criterion indicate that IFABP-PX60
is 200-fold more efficient than free PX. The results described
here indicate that a host-guest system based on a fatty acid
binding protein can be used to generate systems that mimic three
key features of enzymatic catalysis: selectivity, rate enhancement,
and turnover. Efforts to introduce additional functionality using
site-directed mutagenesis to increase the rate of reaction are
currently in progress.
amino acid added
turnovers (n)
L-Glu/D-Glu
ee (%)
none
>0.99
5.3 ( 0.03
5.7 ( 0.27
8.5 ( 0.14
9.0 ( 0.60
5.1 ( 0.12
28 ( 0.10
28 ( 0.11
4.7 ( 0.18
21 ( 0.24
24 ( 0.16
32 ( 0.09
68 (L)
70 (L)
79 (L)
80 (L)
67 (L)
93 (L)
93 (L)
65 (L)
91 (L)
92 (L)
94 (L)
Ala (5a)
Val (5b)
Leu (5c)
Pgl (5d)
Phe (5e)
Tyr (5f)
D-Tyr (5f)
L-Tyr (5f)
Trp (5g)
Dopa (5h)
1.3 ( 0.05
1.1 ( 0.03
1.6 ( 0.11
1.1 ( 0.01
3.9 ( 0.15
4.2 ( 0.07
0.94 ( 0.04
3.1 ( 0.09
2.2 ( 0.04
4.3 ( 0.12
results of these experiments are summarized in Table 1. Initial
experiments with amino acids bearing alkyl side chains including
Ala (5a), Val (5b), and Leu (5c) did produce a small amount of
turnover. Addition of Leu produced the highest levels of Glu
(1.6 turnovers in 24 h). More significant levels of turnover were
obtained using amino acids possessing aromatic side chains. The
addition of Phe (5e), Tyr (5f), Trp (5g), and Dopa (5h) all gave
several turnovers (2.2-4.3) in 24 h. This back reaction appears
to be stereospecific, since substitution of L-Tyr with D-Tyr results
in no conversion beyond what is observed under single turnover
conditions. The enantioselectivities obtained in these catalytic
reactions are of particular interest. The use of aromatic amino
acids as amine sources gives excellent enantioselectivities. Table
1 shows values ranging from 91 to 94% ee. These results are
quite different than the lower selectivities obtained under single
turnover conditions. A possible explanation for this behavior is
that IFABP-PX60 possesses racemase activity. Under single
turnover conditions, the amino acid product (Glu, 4) can remain
bound to the protein where racemization can occur. However,
under catalytic conditions, the amino acid used as the amine
source can displace the bound amino acid product thus preventing
racemization. This model is consistent with the correlation we
observe between rate and enantioselectivity. With the alkyl-
substituted amino acids, low levels of turnover and low values
of ee were obtained; with aromatic amino acids, increased turnover
and higher enantioselectivity were observed. Finally, longer
incubation times with Phe or Tyr result in continued reaction and
many turnovers. We have monitored the IFABP-PX60-catalyzed
production of Glu for up to 14 days at which time over 50
Acknowledgment. The authors thank S. Flaim for modeling IFABP-
PX60, Dr. C. Frieden for providing E. coli MG1655/pIFABP-V60C, M.
Brown, C. Lambert, and Dr. R. Davies for assistance with the protein
purification, and D. Qi for performing the experiments with metal ions.
This work was supported by grants from the National Science Foundation
(NSF-CHE-9506793) and the Petroleum Research Fund (PRF#28140-
G4).
Supporting Information Available: Descriptions of molecular
modeling and the procedures used to prepare, characterize, and study
the reactions catalyzed by IFABP-PX60 (7 pages). See any current
masthead page for ordering and Internet access instructions.
JA972771H
(12) For pyridoxamine, KM probably represents the dissociation constant
for the ketimine complex. For IFABP-PX60, KM may include both covalent
and noncovalent binding terms.