Selective stabilization of the chorismate mutase transition state by a positively charged hydrogen bond donor

Article abstract of DOI:10.1021/ja0341992

Citrulline was incorporated via chemical semisynthesis at position 90 in the active site of the AroH chorismate mutase from Bacillus subtilis. The wild-type arginine at this position makes hydrogen-bonding interactions with the ether oxygen of chorismate.

Full text of DOI:10.1021/ja0341992

Published on Web 02/22/2003
Selective Stabilization of the Chorismate Mutase Transition State by a
Positively Charged Hydrogen Bond Donor
Alexander Kienh o¨ fer, Peter Kast, and Donald Hilvert*
Laboratorium f u¨ r Organische Chemie, Swiss Federal Institute of Technology, ETH H o¨ nggerberg,
CH-8093 Z u¨ rich, Switzerland
Received January 16, 2003 ; E-mail: hilvert@org.chem.ethz.ch
Chorismate mutase (CM) catalyzes the Claisen rearrangement
of (-)-chorismate (1) to prephenate (3), the first committed step
1
in the biosynthesis of phenylalanine and tyrosine. Despite extensive
experimental and theoretical work, its mechanism of action remains
controversial.
Figure 1. Schematic view of the BsCM active site with bound transition
4
state analogue (compound 4, red). In the wild-type enzyme the blue residue
at position 90 is arginine (X ) NH2 ) and in the Arg90Cit variant it is
citrulline (X ) O).
The enzymatic reaction, like its uncatalyzed counterpart, proceeds
+
2
via a chairlike pericyclic transition state (2) in which C-O cleavage
3
precedes C-C bond formation. Electrostatic stabilization of this
Table 1. Kinetic Parameters of BsCM Variantsa
highly polarized species by a positively charged residue, either an
arginine or a lysine, positioned next to the ether oxygen of the
breaking C-O bond has been suggested by crystallographic and
mutagenesis studies of the structurally diverse Bacillus subtilis
k
cat
K
(µM)
m
k
cat/K
m
K
i
enzyme
BsCM*
(s-1)
46
0.0026 270 9.6
(M-1 s-1
)
k
cat/kuncat
(µM)
5
6
98 4.7 × 10
23 04 .0 × 10
1.2
6.8
BsCM),4 Escherichia coli (EcCM), and yeast (ScCM) mutases.
-6
7,8
9
Arg90Cit BsCM*
(
10
This hypothesis has found further support in QM/MM calculations.
a
6
The enzymes were assayed as previously described at 30 °C in 50
mM phosphate buffer (pH 7.5). Ki values were obtained by standard
inhibition assays with 4.6 Errors on all parameters were less than 15%.
BsCM* is the semisynthetic B. subtilis chorismate mutase containing the
D102E mutation. The recombinant BsCM* protein measured under identical
However, more recent computational studies indicate that the
cationic group stabilizes chorismate in the ground and transition
state to similar extents.11 It has even been argued that preferential
,17
transition state binding is unimportant for chorismate mutase
activity.12 Instead, the efficiency of the enzyme has been attributed
mainly to conformational restriction of the substrate in such a way
-1
conditions gave kcat ) 49 ( 2 s and Km ) 105 ( 12 µM, in good
agreement with the published kinetic parameters for wild-type BsCM.
6,18
It was inhibited by 4 with a Ki of 1.0 ( 0.2 µM.
12,13
as to confine the reactive centers to contact distances.
The critical cationic residue in BsCM is Arg90 (Figure 1). Even
conservative substitution of this amino acid with a positively
charged lysine leads to substantial reductions in catalytic efficacy.5
However, the mutations that have been investigated to date have
not been isosteric with arginine and are typically accompanied by
2-mercaptoethane sulfonate to give the BsCM(1-87) fragment as
a thioester. The C-terminal fragments, corresponding to residues
88-127 with either arginine or citrulline at position 90, were
prepared by solid phase peptide synthesis using standard Fmoc
protocols. Asp102 in these peptides was replaced by glutamate to
avoid problems with aspartimide formation. Control experiments
with recombinant D102E BsCM (BsCM*) confirmed that this
mutation does not significantly alter the catalytic properties of the
enzyme (Table 1). Following cleavage from the solid support, the
synthetic BsCM(88-127)* fragments were purified by HPLC and
separately coupled with the BsCM(1-87) thioester. Chemical
ligations were performed with ca. 1 mM of each peptide in 100
mM Tris-HCl buffer (pH 8) containing 6 M guanidinium chloride
and 2.5% thiophenol for 24 h. The ligated polypeptides, BsCM*
and Arg90Cit BsCM*, were subsequently folded by 100-fold
dilution in 50 mM glycine buffer (pH 8.9) containing 5% 2-propanol
and 10% glycerol. After concentration, the folded proteins were
purified by ion-exchange chromatography on a MonoQ column. A
separate dedicated column was used for the Arg90Cit variant to
preclude contamination by another chorismate mutase.
,6
m
large increases in the K value for chorismate, making it difficult
to distinguish unambiguously between ground state and transition
state effects. To overcome this problem, we have prepared a BsCM
variant containing citrulline, an isosteric but neutral arginine
analogue, at position 90. This minimal substitution of a charged
with a neutral hydrogen bond donor allows the importance of the
positive charge in the differential stabilization of the ground and
transition states to be assessed directly.
BsCM and the Arg90Cit variant were prepared by a native
chemical ligation strategy,14 exploiting Cys88 to mediate segment
condensation. The N-terminal peptide fragment corresponding to
residues 1-87 was biosynthesized in E. coli strain KA13 as a fusion
with the Mxe GyrA intein and a chitin-binding domain.15 KA13
has deletions of both endogenous E. coli chorismate mutase genes,16
so that contamination from chromosomally encoded chorismate
mutases can be excluded. The fusion protein was purified on a chitin
column and the intein splicing intermediate was captured with
The semisynthetic proteins were analyzed by electrospray
ionization mass spectroscopy (ESI-MS) and shown to have the
3206
9
J. AM. CHEM. SOC. 2003, 125, 3206-3207
10.1021/ja0341992 CCC: $25.00 © 2003 American Chemical Society

C O MMU N I C A T I O N S
expected mass (BsCM*: found 14 502 ( 2 daltons, expected
4 503 daltons; Arg90Cit BsCM*: found 14 503 ( 2 daltons,
bond donor involved in selective stabilization of chorismate in the
transition state. It is noteworthy that structurally unrelated AroQ
1
7
9
expected 14 504 daltons). Their circular dichroism spectra are
superimposable on that of the recombinant enzyme purified under
native conditions, indicating identical overall secondary structure.
Like recombinant BsCM, they are homotrimeric in solution, eluting
as a single peak with identical retention times from a Superose 12
size-exclusion column. Their trimeric quaternary structure was
additionally confirmed by sedimentation velocity ultracentrifugation,
which yielded average molecular masses of 41 500 and 43 600
daltons for semisynthetic BsCM* and for the citrulline variant,
respectively, in good agreement with the expected mass for a trimer
of 43 509 daltons.
mutases such as EcCM and ScCM have a similarly positioned
cation, albeit a lysine rather than an arginine, whereas the relatively
2
2
inefficient catalytic antibody 1F7 lacks this feature. Efficient
catalysis of the chorismate mutase rearrangement evidently requires
more than an active site that is simply complementary in shape to
the reactive substrate conformer;12 electrostatic stabilization of the
polarized transition state appears to be paramount.
Acknowledgment. We thank Stefan van Sint Fiet for preparing
the D102E mutant, Angelo Guainazzi for optimization of the BsCM-
(1-87) thioester, Dr. Rosalino Pulido for the synthesis of 4, and
the Swiss National Foundation, the ETH Z u¨ rich and Novartis
Pharma for generous support of this work.
Semisynthetic BsCM* is fully active as a catalyst, affording kcat
and K
are indistinguishable from those of its recombinant counterpart
Table 1). The Arg90Cit variant is also active, albeit substantially
m
parameters for the chorismate mutase rearrangement that
(
Supporting Information Available: Experimental details for the
synthesis and characterization of BsCM* and Arg90Cit BsCM*. This
material is available free of charge via the Internet at http://pubs.acs.org.
less so than the wild-type enzyme. Replacement of arginine by
4
citrulline causes a >10 -fold decrease in kcat and a more modest
2
.7-fold increase in the K
small change in K suggests only minor perturbation of the ground-
state Michaelis complex, although the nonequality of K and the
m
value for chorismate (Table 1). The
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m
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(
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5,6
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JA0341992
J. AM. CHEM. SOC.
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