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P.H. Lodha et al. / Biochimica et Biophysica Acta 1804 (2010) 1424–1431
ytCBS-N84A (Table 1), no β-elimination or substrate-induced inacti-
vation are detected. Additionally, the fluorescence resonance energy
transfer from tryptophan residue(s) to the internal aldimine and the
external aldimine of aminoacrylate forms of the cofactor is similar to
that of the wild-type enzyme (Fig. 5). This suggests that, despite the
loss of the hydrogen bond to O3’ of the cofactor, the orientation of PLP
in the internal aldimine and the external aldimine of aminoacrylate, as
well as the equilibrium between the open and closed conformations of
the active site, of the N84A mutant is similar to that of the wild-type
enzyme (Fig. 5). Therefore, while elimination of the hydrogen bond
between PLP-O3' and N84 of ytCBS likely alters the movement and
orientation of the cofactor during the catalytic cycle, the change is
subtle. The observed change in the kinetic parameters of ytCBS-N84A
may also be due to repositioning of the backbone of this residue as a
result of elimination of the tethering hydrogen bonds of the N84 side
chain. This could cause the weakening or loss of the proposed
hydrogen bond between the α-carboxylate of the substrate and the
backbone NH moiety of N84 [33].
elimination side reaction activity, the mutant is uniquely inactivated
because it lacks the ability to efficiently release aminoacrylate from
the active site [14]. In contrast with stTrpS-βQ114N, the carboxylate
moiety of the aspartate side chain of the ytCBS-N84D mutant may
stabilize the developing positive charge of the enamine nitrogen
during the attack of aminoacrylate on C4' of the PLP cofactor, thereby
facilitating the inactivation of the enzyme.
While a β-elimination activity has been observed for 10 (T81A,
S82A, N84D, N84H, T85A, Q157A, Q157E, Q157H, Y158F and S289) of
the 20 site-directed mutants, targeting 13 active-site residues (Fig. 6),
reported for ytCBS, aminoacrylate-mediated inactivation has been
reported for only three (N84D, Q157H and Y158F) [15,16,35]. PLP
enzymes catalyzing β-replacement reactions have evolved to facili-
tate the transaldimination reaction with substrates and products, but
not aminoacrylate, thereby minimizing the β-elimination side
reaction, which produces pyruvate and ammonia, and inactivation
by aminoacrylate. The ability to selectively facilitate the transaldimi-
nation of substrates and products and diminish the corresponding
reaction with aminoacrylate is likely a function of conformational
constraints imposed by the enzyme [14]. The data presented
demonstrate that residue N84 of ytCBS is a determinant of cofactor
positioning and of reaction specificity, via its role in the regulation of
aminoacrylate partitioning to favor the β-replacement reaction over
the release of aminoacrylate, via β-elimination.
The histidine and aspartate substitutions of ytCBS-N84 were selected
to modify the capacity of this residue to form hydrogen bonds. The 640
and 100-fold reductions in the kcatF/KLm-SFer and kcatR/KLm-CRth of the β-
replacement and reverse-physiological activities of N84H are similar to
those observed for N84A (Table 1). However, a change in the orientation
of the PLP cofactor within the active site, compared to the wild-type
enzyme and ytCBS-N84A, are observed for N84H (Fig. 5). A marginal β-
elimination activity (kcatE/Km-SEer=0.34 0.06 M-1 s-1) is also detected
L
Acknowledgements
for ytCBS-N84H, suggesting a corresponding change in active-site
dynamics.
The authors thank the reviewers of this paper for their insightful
comments and suggestions. This work was supported by a grant from
the Natural Sciences and Engineering Research Council of Canada.
The kcatF/KmL-SFer and kcatR/KLm-CRth of N84D are reduced by 3 and 4
orders of magnitude, respectively. This drastic reduction in catalytic
efficiency, compared to the wild-type enzyme and the N84A and
N84H mutants, the 3-fold reduction in PLP-saturation, the lack of
accumulation of an aminoacrylate intermediate (Figs. 1 and 2) and the
observed inactivation of this mutant demonstrate that the cofactor is
not bound in a productive orientation and that the equilibrium
between the open and closed conformations of the active site is
disrupted. The side chain Oδ1 and Nδ2 atoms of hCBS-N149, which
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