is shown in ball-and-stick representation. The picture was drawn with PyMol (ver, 0.98-2005 DeLano
Scientific LLC) using PDB entry 1C7N.
The analysis of the spectral properties of recombinant cystalysin reveals that the native
enzyme exhibits two absorption bands in the visible region at 418 and 320 nm, whose
intensity is dependent on pH (Figure 3). Titration of enzyme-bound coenzyme in the pH
range 5.9-9.7 is consistent with a single deprotonation event with a pK value of about 8.4.
However, the absorbance spectra do not show the complete conversion of the 418 nm band
into the 320 nm band, thus suggesting the involvement of multiple species. On the basis of
their fluorescence properties, the 418 nm band, predominating at low pH, has been attributed
Cystalysin: An Example of the Catalytic Versatility… 103
to the internal Schiff base in the ketoenamine form, while the 320 nm band, predominating at
high pH, has been attributed to a substituted aldamine which forms upon addition of a
deprotonated nucleophile or a hydroxyl group to the imine double bond [15]. Altogether, the
spectral changes of cystalysin as a function of pH have been interpreted according to the
model shown in Figure 4. It involves the interconversion between XH-I and X-
ketoenamine forms absorbing at 418 nm and protonated (II) and unprotonated (IV)
substituted aldamine forms absorbing at 320 nm. At low pH (pH<8.4) I and II are present,
while at high pH (pH>8.4) III and IV are present. XH is the group performing the
nucleophilic attack on the C4’ of the internal aldimine and its deprotonated X- form is the
more favorable for forming the adduct. Site-directed mutagenesis experiments strongly
well as between III and IV are governed by the aldamine formation. Instead, the equilibrium
between I and III and that between II and IV are governed by a deprotonation/protonation
event. Accordingly, the spectral pK of 8.4 would reflect the ionization of the XH group
whose ionization influence the equilibrium between the species absorbing at 418 and 320 nm.
Figure 3. Absorbance spectra of 50 µM cystalysin in 20 mM Bis-Tris propane at pH 5.9 (⎯) and pH
α,β-ELIMINATION IS THE MAIN REACTION OF CYSTALYSIN
pyruvate [17]. This allows to outline the catalytic mechanism for the desulphydrase reaction
shown in Figure 5. Upon binding of the substrate L-cysteine, the Michaelis complex I is
rapidly converted to the external aldimine II. Then, the abstraction of the Cα-proton of the
substrate produces a carbanionic intermediate that is stabilized as the characteristic quinonoid
intermediate (III) and the subsequent elimination of H2S generates the PLP derivative of the
aminoacrylate (IV). Finally, a reverse transaldimination takes place forming iminopropionate
104 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
and regenerating the internal aldimine. The reaction end product iminoproprionate is released
and hydrolyzed to pyruvate and ammonia outside the active site [14].
Figure 4. Structures of the coenzyme form in cystalysin as a function of pH.
Table 1. Kinetic parameters for the α,β-elimination of various substrates catalyzed by
cystalysin in 20 mM potassium phosphate buffer pH 7.4 at 25°C
kcat (s-1) Km (mM) kcat/ Km (mM-1s
L-cysteine 11.4 ± 0.3 0.63 ± 0.11 18 ± 3
L-cystathionine 13.03 ± 0.7 1.38 ± 0.2 9.4 ± 1.4
L-cystinea 21.1 ± 0.3 0.68 ± 0.05 31 ± 2
L-djenkolic acid 72.2 ± 6.7 0.99 ± 0.15 73 ± 13
L-serine 0.36 ± 0.02 6.92 ± 1.15 0.052 ± 0.009
Β-chloro-L-alanine 59.9 ± 2.3 1.21 ± 0.15 50 ± 6
O-acetyl-L-serine 63.3 ± 3.0 1.6 ± 0.2 40 ± 5 a
The study of the reaction of cystalysin with various sulfur- and non-sulfur-containing
amino acids, as well as with disulfidic amino acids, has shown the relatively broad substrate
specificity of cystalysin. Structural elements of the substrate molecule playing a critical role
in the catalytic efficiency of cystalysin-catalyzed α,β-elimination are a second cysteinyl
Cystalysin: An Example of the Catalytic Versatility… 105
seem to be a cysteine desulphydrase, as previously claimed [11], but should more properly be
considered as a cyst(e)ine C-S lyase.
Figure 5. Catalytic mechanism for the α,β-elimination of L-cysteine catalyzed by cystalysin.
Several reaction intermediates of the α,β-elimination reaction catalyzed by cystalysin
have been identified by studying the interaction of the enzyme with both substrates or
substrate analogs. Among the substrates, the interaction of cystalysin with L-serine, analyzed
by conventional spectroscopy, allows only the detection of a band absorbing at 429 nm
attributed to the external aldimine (Figure 6A). Likewise, the interaction of the enzyme with
β-chloro-L-alanine, studied by UV-vis stopped-flow spectroscopy, leads to the formation of a
band absorbing at 330 nm which has been attributed to an external aldimine in the enolimine
form (Figure 6B). Other reaction intermediates have not been identified so far in the catalytic
106 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
been suggested that glycine, due to the absence of side-chain carbon atoms, could be unable
to maintain the scissile bond parallel to the aldimine p orbitals, thus preventing the
subsequent hydrogen abstraction [15].
analogs. (A) Absorption spectra of 6.5 µM cystalysin (⎯) and immediately after addiction of 100 mM
addition of 10 mM β-chloro-L-alanine (⎯). (C) Absorption spectra of 7 µM cystalysin (⎯), and in the
presence of 20 mM glycine(----), 494 mM L-homoserine (······) and 204 mM L-methionine (- · -). In
each case the buffer was 20 mM potassium phosphate pH 7.4.
Cystalysin: An Example of the Catalytic Versatility… 107
Extensive investigations recently undertaken on the kinetic features of cystalysin have
allowed the identification of residues involved in catalysis and have provided new insights on
the catalytic mechanism of the enzyme.
with a pK of 6-6.4 is involved in catalysis and must be unprotonated to achieve maximum
substrate [16]; ii) a group with a pK of ~8 affects the kcat of the reaction and must be
unprotonated to achieve maximum velocity. However, as the kcat differs by a factor of only 4-
5 at low and high pH, it was suggested that this group influences the chemistry of the reaction
even if it is not directly involved in catalysis. As proposed, this pK may reflect either the
ionization of the coenzyme phosphate group or the ionization of an unknown group which
induces a conformational change resulting in the conversion from a less to a more
catalitically competent conformation.
Site-directed mutagenesis studies have indicated that Lys 238, the residue which forms
the internal aldimine, is essential for the α,β-elimination reaction catalyzed by cystalysin. In
particular, mutant enzymes in which Lys 238 has been replaced by alanine (K238A) or
and decay of the Schiff base species has been significantly decreased in the mutants K238A
and K238R. Kinetic studies indicate that K238A mutant is inactive in the α,β-elimination
reaction, while the K238R retains poor eliminase activity. In addition, the analysis of the
reaction of Lys 238-mutants with L-methionine and L-homoserine shows that mutation of the
active site lysine to arginine does not prevent the Cα-hydrogen abstraction leading to the
quinonoid intermediate. On the other hand, mutation of Lys 238 to alanine seems to block the
reaction at the step of the external aldimine. All together, these results led to the proposal that
Lys 238 in cystalysin fulfills a triple role: it strengthens the PLP binding; it enhances the
formation and dissociation of the enzyme and ligand Schiff bases, allowing an easier
transaldimination; it might also have an essential catalytic role, possibly participating in the
reaction as a general base abstracting the Cα-proton from the substrate, and a general acid
protonating the β-leaving group [18].
Numerous insights on the kinetic features of cystalysin have been obtained by studying
changed to alanine. The results indicate that Tyr 64 plays a role in cofactor binding but is not
essential for catalysis, as its mutation results in only about 90% reduction in the kcat and
kcat/Km values with respect to wild-type. However, stopped-flow analyses of the interaction of
rapid chemical quench studies demonstrate that Tyr64 mutation changes the rate-limiting step
of the α,β-elimination reaction. In fact, α-aminoacrylate formation is rate-determining in the
108 Barbara Cellini, Riccardo Montioli and Carla Borri Voltattorni
mutant while the rate-limiting step in the α,β-elimination catalyzed by wild-type cystalysin is
most probably associated to product release. On the basis of structural data it has been
proposed that Tyr64 during the catalytic cycle could act by correctly positioning the Lys 238
ε-amino group toward the leaving group to facilitate catalysis [16]. The recent insights on the
proposed α,β-elimination reaction mechanism catalyzed by cystalysin are highlighted in
Figure 7. Proposed role of Lys 238 and Tyr 64 in the α,β-elimination reaction mechanism catalyzed by
THE ALANINE RACEMASE AND TRANSAMINASE ACTIVITIES
Although optimized for catalyzing the α,β-elimination, the active site structure of
cystalysin contains structural elements required for the catalysis of other reactions typical of
PLP-enzymes. In particular, Lys 238, the PLP-binding lysine, is located on the si face of PLP,
while Tyr 123 and Tyr 124 are located on the re face of the cofactor. These active-site
residues are properly positioned to act as acid-base catalysts for the pro-S and pro-R proton
abstraction from an appropriate substrate. A similar active site architecture has been observed
in alanine racemase from Bacillus stearothermophilus [19], a protein belonging to Fold type
III group of PLP-dependent enzymes, which occurs ubiquitously in eubacteria and catalyzes
the interconversion of L- and D-alanine. As shown in Figure 8, the comparison of the active
site of cystalysin and alanine racemase reveals a similar arrangement of the acid-base
catalysts even if in alanine racemase Tyr 265 is located on the si face of PLP while Lys 39 is
located on the re face [19]. On the basis of the crystal structure of the complex of alanine
abstracts the α-proton from the substrate, and a second acid-base catalyst which reprotonates
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