Synthesis of aspartame by thermolysin: An x-ray structural study

Article abstract of DOI:10.1021/ml500101z

Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the artificial sweetener aspartame, a multiton peptide synthesis catalyzed by the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a crystal, rationalize the reaction's substrate preferences and reveal an unexpected form of substrate inhibition that explains its sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages.

Full text of DOI:10.1021/ml500101z

Letter
pubs.acs.org/acsmedchemlett
Synthesis of Aspartame by Thermolysin: An X‑ray Structural Study
Gabriel Birrane,*,† Balaji Bhyravbhatla,‡ and Manuel A. Navia*,§,∥
†Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215, United States
‡Xtal BioStructures, 12 Michigan Drive, Natick, Massachusetts 01760, United States
§Altus Pharmaceuticals, Waltham, Massachusetts 02451, United States
S
* Supporting Information
ABSTRACT: Protease mediated peptide synthesis (PMPS)
was first described in the 1930s but remains underexploited
today. In most PMPS, the reaction equilibrium is shifted
toward synthesis by the aqueous insolubility of product
generated. Substrates and proteases are selected by trial and
error, yields are modest, and reaction times are slow. Once
implemented, however, PMPS reactions can be simple,
environmentally benign, and readily scalable to a commercial
level. We examined the PMPS of a precursor of the artificial
sweetener aspartame, a multiton peptide synthesis catalyzed by
the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a
crystal, rationalize the reaction’s substrate preferences and reveal an unexpected form of substrate inhibition that explains its
sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial
peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages.
KEYWORDS: Protease mediated peptide synthesis, aspartame, crystallography, thermolysin
racemic mixture of DL-phenylalanine methyl ester. In contrast,
ergmann and Fraenkel−Conrat reported the first protease-
1
Bmediated synthesis of a peptide bond in 1938. Their although both enantiomers of ZA can bind to TLN, only
carbobenzoxy-L-aspartic acid is used in practice since
carbobenzoxy-D-aspartic acid inhibits the enzyme.10
elegantly simple procedure, catalyzed by the enzyme papain, led
to a precipitate that provided a 22% yield of benzoyl-L-leucyl-L-
leucine anilide but required a reaction time of 2 days. Protease-
mediated peptide condensation reactions subsequently de-
scribed in the literature2 have been hindered similarly by
excessive reaction times, trial and error manipulation of
proteases and reaction conditions, and characteristically poor
binding affinity of the unnatural, modified substrates used. As
such, it has been difficult to exploit this approach. However,
once a reaction is optimized,3 its relative ease of scalability and
benign environmental impact make PMPS commercially
attractive in spite of its shortcomings, when compared to
other large-scale peptide synthesis strategies.4,5
Aspartame (APM) is a protected dipeptide (L-aspartyl-L-
phenylalanine methyl ester) that is widely used as a low-calorie
sweetener.6 More than 19 000 metric tons of APM are
produced per year,7 making it the most highly synthesized
peptide in the world. Over 2000 metric tons of the annual
output of APM is made enzymatically,8 using the protease
thermolysin (TLN) to catalyze the condensation of the chiral
aspartame-precursor, carbobenzoxy-L-aspartyl-L-phenylalanine
methyl ester (ZAPM), from the protected amino acid
substrates carbobenzoxy-L-aspartic acid (ZA) and L-phenyl-
alanine methyl ester (PM).3,7−9 Deprotection of ZAPM by
catalytic hydrogenation yields aspartame as the final product.3,9
In the ZAPM precursor reaction, TLN is enantioselective for
the desired L-phenylalanine methyl ester substrate from a
Isowa and collaborators discovered the synthesis of ZAPM
by TLN after a systematic evaluation of the enzyme-mediated
coupling of PM to a series of N-protected L-aspartic acid
analogues.3 Subsequently, a mechanism of action for the TLN-
mediated synthesis of ZAPM in aqueous solvent was proposed
by Oyama et al.;10 this accounts for the rate saturation of the
reaction observed with increasing concentration of the carboxy-
donor substrate ZA and the linear rate increase of the reaction
within the concentration range explored for the amino-donor
substrate PM. These results were confirmed by Wayne and
Fruton11 and extended to biphasic aqueous−organic solvent
mixtures12 and organic solvents13 by Nakanishi et al. The
enzyme data obtained by these groups, summarized in Table 1,
illustrate the poor binding of the substrates to thermolysin and
the sluggishness with which the reaction proceeds. These
characteristics make it difficult to study the reaction using
conventional steady-state kinetic methods.
The TLN used in the synthesis of ZAPM is a heat stable
extracellular Zn2+ metalloprotease (EC 3.4.24.4) that is readily
isolated by direct crystallization from microbial extracts of
Bacillus thermoproteolyticus.14 As such, it is both plentiful and
Received: March 10, 2014
Accepted: April 10, 2014
Published: April 10, 2014
© 2014 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Table 1. Enzyme Parameters for the Synthesis of ZAPM by
TLN As Obtained from the Literature
solvent
system
Km (mM) Km (mM)
kcat/Km
ref
ZA
PM
(mM−1 min−1
)
a
Oyama et al.10
aqueous
n/d
300
10
10
0.160
0.160
a
Wayne and
aqueous
Fruton11
Nakanishi12
biphasic
250
2.8
0.142
b
aq/org
a
The parameters reported by Oyama et al.10 and Wayne and Fruton11
were derived from a modified Michaelis−Menten model applied to
b
data collected in aqueous solvent. Nakanishi et al.12 derived their
enzyme parameters from data measured in a biphasic aqueous/organic
solvent mixture. At the higher end of the ZA concentration range
explored, a slight rate reduction vs ZA concentration was observed.12
inexpensive. High quality X-ray structures of TLN are available,
in complex with both substrates15−17 and inhibitors.18−20 These
provide a firm basis for understanding the mechanism of
peptide hydrolysis used by the enzyme,17,21 as well as the
probable geometry of substrate binding.20
Here, we report the X-ray structures of TLN in complex with
the amino- and carboxy-donor substrates PM and ZA
individually, as well as with both substrates together. Earlier
studies22,23 with cross-linked microcrystalline TLN enzyme
suspensions24 showed that the latter conditions would lead to
the synthesis of a ZAPM precipitate, whose chemical analysis
has been described in detail.23 On the basis of our new
crystallographic data (Table S1 in the Supporting Information)
and the existing literature (Table 1), we propose a mechanism
of action for the synthesis of ZAPM by TLN that explains the
sluggish reaction rate observed and provides a solid foundation
to improve the efficiency of this commercially important
process.
The crystal structures determined in this study (Table S1 in
the Supporting Information) all show strong ligand electron
density in the TLN active site (Figure 1) that is readily
interpretable in chemical terms. In the structure of the amino-
donor PM in complex with TLN, a PM molecule occupies the
active site of TLN (Figure 1a) in a conformation that is
consistent with that expected for an amino-donor substrate in a
TLN-mediated peptide synthesis reaction. This productive
conformation can be inferred, for example, from the structure
of the amino-donor side (S1′−S2′)25 of the potent (Ki = 0.068
nM) phosphonamidate transition state analogue inhibitor
carbobenzoxy-L-phenylalanine-phosphonamidate-L-leucyl-L-ala-
nine (ZFPLA)26 in its complex with TLN.20 The structures of
ZFPLA and PM in their respective complexes with TLN
(Figure 2a), highlight the close correspondence between the
position and directionality of the phosphonamidate nitrogen of
ZFPLA and the free amino group of PM. Given an
appropriately configured carboxy-donor as a cosubstrate, it
would be structurally reasonable for the amino-donor PM to
participate in the formation of a tetrahedral intermediate within
the ZAPM peptide coupling reaction (Scheme 1). In addition
to the amino donor site, two molecules of PM occupy
interpretable electron density adjacent to a crystal packing
contact between TLN molecules. These molecules are 5.3 Å
away from each other in exposed sites on the surface of the
protein (Figure S1 in the Supporting Information).
Figure 1. Structure of the active site region of thermolysin in complex
with L-phenylalanine methyl ester (PM) and carbobenzoxy-L-aspartic
acid (ZA). σA-weighted 2Fo − Fc electron density maps are contoured
in blue at 1.5σ. Key residues in the active site of TLN14−19 are
identified (green). Nitrogen and oxygen atoms are shown in blue and
red, respectively. The active site zinc atom is purple. (A) PM (pink)
occupies the expected position for an amino-donor substrate in the
peptide coupling reaction.19−21,26 (B) Unexpectedly, the carboxy-
donor ZA (khaki) occupies the same amino-donor site as does PM in
panel a.
bidentate binding of the α-carboxyl of ZA to the TLN active
site zinc closely resembles (Figure 2c) the interaction observed
when TLN interacts with the potent (Ki = 50 nM) inhibitor N-
(1-carboxy-3-phenyl-propyl)-L-leucyl-L-tryptophan (CLT).19
The strong bond between CLT and TLN suggests that the
intrinsic inhibitory potency of ZA vs TLN is likely to be
substantial.
When PM and ZA substrates were both added to a
diffraction quality crystal of TLN in stabilizing solution, a fine
precipitate formed over time in the crystallization vessel,
reminiscent of that formed in the synthesis of ZAPM by a slurry
of cross-linked microcrystalline TLN.23 The resulting structure
(Table S2 in the Supporting Information) showed difference
electron density (data not shown) equivalent to that observed
when the carboxy-donor substrate ZA bound at the amino-
donor site (Figures 2b,c), consistent with the greater relative
binding affinity of ZA for the enzyme (Table 1). The absence of
electron density corresponding to the product ZAPM was
consistent with its aqueous insolubility and precipitation out of
solution, which drives this reaction.3,9−13,23 Additional electron
density adjacent to a crystal packing contact between TLN
molecules corresponds to the two PM molecules identified in
the TLN−PM complex above (Figure S1 in the Supporting
Information). The two PM molecules bind approximately 12
In the structure of the carboxy-donor ZA in complex with
TLN (Figure 1b), ZA unexpectedly occupies the same binding
site as the amino-donor substrate PM (Figure 2b). The
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ACS Medicinal Chemistry Letters
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Scheme 1. Proposed Mechanism of Action for the
Thermolysin-Catalyzed Synthesis of Aspartame Precursor Z-
L-Asp-L-Phe-OMe (ZAPM)
2b) when it interacts with TLN (Figure 1b). Moreover, the
structural similarity between ZA and CLT19 (Ki = 50 nM) in
their TLN complexes (Figure 2c) suggests that the binding
affinity of ZA for the amino-donor site of TLN is likely to be
substantial. This atypical form of substrate inhibition would not
be detectable by steady-state enzyme kinetics alone. Absent the
structural insights presented here, the cryptic competitive
inhibition of PM substrate binding by ZA, would only show
itself as a shift to higher apparent Km and a slower overall
reaction rate (kcat). This profile is consistent with the poor
binding and sluggish performance reported10−12 (Table 1) for
the TLN-mediated synthesis of ZAPM.
A further complication in this reaction is possible if, after
bound ZA transfers to the carboxy-donor site of TLN (as in
Figure 3), a second ZA molecule (ZA*) were to bind to the
amino-donor site in competition with PM. This form of
substrate inhibition27 would manifest as a decrease in the
steady-state rate of reaction at higher concentrations of ZA,
which is consistent with the reaction inhibition profile reported
by Nakanishi et al.12
In its conventional hydrolysis of peptides and proteins, TLN
is modestly specific for residues like Ile, Leu, and Phe on the
amino-donor (S1′) side of the scissile bond but will accept a
broad spectrum of residues on the carboxy-donor (S1−S2)
side.19,21,28 This is consistent with the depth and texture of the
S1′ specificity pocket (into which both PM and ZA bind
preferentially; Figure 2b) and the contrasting lack of distinctive
structural features on the enzyme surface corresponding to S1
and S2 on the carboxy-donor side (Figure 3). Tight, specific
binding to the S1′ site alone is probably sufficient to properly
orient a natural protein or extended peptide substrate for
cleavage by TLN. However, in an unnatural peptide synthesis
Figure 2. Binding of substrates and inhibitors to the active site of
thermolysin. Residues E143, Y157, and H231 (green) represent the
catalytic machinery of TLN, along with the active site zinc ion, which
is held in place by residues H142, H146, and E166 (green). (A)
Structure of PM (pink), superimposed on that of the transition state
analogue inhibitor ZFPLA20,26 (PDB ID 4TMN, brown) after
alignment of the TLN protein coordinates of the corresponding
complex structures. The arrow highlights the close positional and
orientational similarity between the amino group of PM and the
phosphonamidate nitrogen of ZFPLA. (B) Structure of PM (pink)
superimposed on that of ZA (khaki) shows how both ligands occupy
the same substrate amino-donor site on the TLN enzyme. (C)
Structure of ZA (khaki) superimposed on that of the TLN inhibitor
CLT19,21 (PDB ID 1THL, gray). The α-carboxyl of ZA and the
carboxyl of CLT both chelate the catalytic zinc ion of the enzyme in a
bidentate inhibitory conformation (arrow).
and 16 Å away from the ZA molecules in the active site (Figure
S2 in the Supporting Information).
Contrary to expectation, the carboxy-donor substrate ZA
binds preferentially to the amino-donor site of enzyme (Figure
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ACS Medicinal Chemistry Letters
Letter
of the product ZAPM, whose precipitation as the water-
insoluble Phe-OMe salt drives the overall reaction in the
direction of peptide synthesis.
In spite of their commercial importance, potentially broad
applicability and technical simplicity, protease-catalyzed peptide
synthesis reactions have not been adequately characterized,
optimized, or broadly accepted in the practice of modern, large-
scale, solution enzymology. The crystal structures described
here highlight the degree of complexity that can accompany
such unnatural reactions in which experimental conditions are
artificially manipulated to achieve an economically viable, albeit
enzymatically inefficient, result. These structures may also help
to improve the aspartame precursor synthesis reaction directly,
either by guiding the rational selection of alternate substrates,
or by inferring modifications of the TLN enzyme (or other
closely related enzymes)30 for increased reaction efficiency.
More broadly, these results lead one to consider the extent to
which the inefficiency (and unpredictability) associated with
this approach to peptide synthesis may be attributed to
similarly encrypted substrate inhibition that might be
correctable, once identified by more powerful methods like
X-ray crystallography. Fortunately, many of the enzymes that
have been used in the protease-mediated peptide syntheses
documented in the literature2 have themselves been the subject
of extensive crystallographic study,31 and these data may help
predict how the enzymes will interact with various substrates.
To prioritize the reactions that would benefit most from
additional analysis by X-ray crystallography, equilibrium
dialysis, or other direct ligand binding studies could be used
to screen suboptimal peptide synthesis approaches, in the hope
of improving the efficiency, and expanding the commercial
applicability of protease-mediated peptide synthesis reactions.
Figure 3. Stereo view of the active site of thermolysin. PM and ZA are
shown bound to the amino-donor S1′ and carboxy-donor S1−S2 sites
of the enzyme, respectively, in a hypothetical productive conformation
for the TLN-catalyzed condensation of the ZAPM dipeptide product.
PM binding is as shown in Figure 2a. In turn, the productive
conformation of ZA is modeled using the atom-for-atom topological
(though not chemical) equivalence between ZA and the C-terminal
half of the transition state analogue inhibitor ZFPLA. The structure of
ZFPLA in the active site of TLN20 is shown in thin bonds colored
brown and immediately suggests a structure for the transition state
leading to the synthesis of the ZAPM product. All figures were made
using POVSCRIPT29 and ray-traced with POV-Ray (www.povray.
org).
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental procedures, supplementary Table S1 and Figures
S1 and S2. This material is available free of charge via the
Internet at http://pubs.acs.org.
reaction such as TLN-mediated synthesis of ZAPM, the
suboptimal carboxy-donor S1 and S2 substrate-binding site
present a problem since both substrates must be accommo-
dated and properly oriented in a catalytically productive
configuration (as in Figure 3) for the reaction to proceed.
This problem may be resolved in the reaction by channeling the
initial binding of both substrates to TLN through the S1′ site
on the amino-donor side of the active site.
Accession Codes
The atomic coordinates and structure factors have been
deposited in the Protein Data Bank as entries 3QGO, 3QH1,
and 3QH5.
On the basis of the enzyme−substrate complex structures
reported here and the enzyme kinetics data from the literature
summarized in Table 1, we propose a chemically reasonable
mechanism of action for the TLN-catalyzed synthesis of the
aspartame precursor ZAPM, as shown in Scheme 1. We
hypothesize that the reaction is initiated by the preferential
binding of the carboxy-donor substrate ZA to the amino-donor
(S1′) side of the enzyme active site. While still tethered to the
enzyme through its interaction with the catalytic zinc ion, ZA
then rotates out of the amino-donor site to accommodate PM
binding to the enzyme. No steric clashes are evident that would
prevent ZA from sampling a productive carboxy-donor
conformation (Figure 3) as it reorients within the TLN active
site. Conversely, there do not appear to be any compelling
interactions in the poorly defined carboxy-donor site that would
appropriately orient ZA absent its initial binding to zinc. Once
both substrates are productively bound to TLN, the enzyme
can catalyze the formation of a tetrahedral intermediate,
analogous in structure to the inhibitor ZFPLA (Figure 3).
Collapse of the intermediate would then lead to the formation
AUTHOR INFORMATION
Corresponding Authors
*(G.B.) Tel: (617) 667 0025. Fax: (617) 975 5243. E-mail:
gbirrane@bidmc.harvard.edu.
*(M.A.N.) Tel: (781) 389 0686. Fax: (617) 357 7476. E-mail:
mnavia@oxbio.com.
■
Present Address
∥(M.A.N.) Oxford Bioscience Partners, 535 Boylston Street,
Suite 402, Boston, Massachusetts 02116, United States.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Data for this study were measured at beamline X29 of the
National Synchrotron Light Source. Financial support comes
principally from the Offices of Biological and Environmental
Research and of Basic Energy Sciences of the US Department
of Energy, and from the National Center for Research
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ACS Medicinal Chemistry Letters
Letter
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Anne-Laure Dassier for her assistance during data
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Medicine, Beth Israel Deaconess Medical Center, Boston, MA)
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ABBREVIATIONS
■
PMPS, protease mediated peptide synthesis; TLN, thermolysin;
ZA, carbobenzoxy-L-aspartic acid; ZAPM, carbobenzoxy-L-
aspartyl-L-phenylalanine methyl ester; PM, L-phenylalanine
methyl ester; APM, aspartame
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