C O M M U N I C A T I O N S
Cys406Ser mutant to form the reaction intermediate can be
attributed to the removal of the active-site nucleophile. Moreover,
the X-ray structure and modeled arginine show that the Cys406
thiol group is perfectly oriented for attack at the guanidinium carbon
of the L-arginine as it is held in place by H-bonds formed with
several active-site residues. His278 is the probable base.
In summary, the close proximity of the Cys406 thiol to the
guanidinium carbon of the L-arginine substrate, coupled with the
essential role that it plays in the formation of the reaction
intermediate, serves as strong evidence to support the proposed role
of Cys406 in nucleophilic catalysis.15 Nucleophilic catalysis in
arginine deiminase has important implications for the mechanisms
of catalysis in other members of the same enzyme family, which
also conserve the active-site Cys. In addition, the results suggest
that arginine deiminase inhibitor design strategies may be based
on mechanism-based, covalent modification of the active-site
Cys406.
Figure 1. Time courses for the single-turnover reaction of 520 µM arginine
deiminase (wild-type or Cys406Ser mutant) and 10 µM [14C-1]L-arginine
in 50 mM K+Mes/20 mM MgCl2 (pH 5.6) at 25 °C. Reactions were carried
out in a KinTek rapid-quench instrument using 0.6 M HCl as the quench,
CCl4 to precipitate the enzyme, and reversed-phase HPLC to separate the
[14C-1]L-arginine and [14C-1]L-citrulline: (A) (O) [14C-1]L-arginine and (b)
[14C-1]L-citrulline time course data generated with the wild-type enzyme
were fitted with the first-order equations [S]t ) [S]max - ([P]max(1 - e-kt))
and [P]t ) [P]max(1 - e-kt) where “k” is the first-order rate constant; [S]t
and [P]t are the substrate and product concentrations at time “t”. (B) Time
courses for the formation and decay of the 14C-labeled enzyme from the
reaction of wild-type arginine deiminase (blue b) and Cys406Ser arginine
deiminase (green b). The red curve was obtained by fitting the data points
to the kinetic model shown by using the program KINSIM and k1 ) 13
Acknowledgment. This research was supported by NIH Grant
GM28688 to D.D-M and NIH Grant P01 GM57890 to O.H.
References
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(14) Following the submission of this manuscript we have solved the structure
of the Cys406Ala mutant complexed with L-arginine. The new structure
confirms the published model4 and demonstrates that replacement of
Cys406 with Ala does not alter the configuration of active-site residues.
(15) Note in added proof: the X-ray coordinates of the Mycoplasma arginini
arginine deiminase in complex with the product citrulline have been
released at the PDB ID: 1LXY by authors Das, K.; Buttler, G. H.;
Kwiztkowski, V.; Yadav, P.; Arnold, E. This structure reveals a covalent
adduct formed between the active-site Cys and the citrulline carbonyl
carbon, consistent with nucleophilic addition of the Cys at the electrophilic
center.
The X-ray structure of the arginine deiminase active site (see
Figure 2) suggests that replacement of Cys406 with a Ser residue
will not perturb the active-site residues.14 Thus, failure of the
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