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Abstract
The interaction of the enzyme Escherichia coli RI methyl transferase (methylase) with
an arsenic(III) derivative of cacodylic acid has been investigated by optical detection
of triplet-state magnetic resonance (ODMR) spectroscopy in zero applied magnetic field.
The reactive derivative (CH3)2AsSR is formed by the reduction of cacodylate by a thiol.
The As(III) derivative binds to the enzyme by mercaptide exchange with a cysteine
(Cys) residue located close to a tryptophan (Trp) site. The arsenical binding selectively
induces an external heavy-atom effect, perturbing the nearby Trp residue in the enzyme.
Zero-field splittings (ZFS) and total decay rate constants of the individual triplet-state
sublevels of the Trp residue in the presence and absence of perturbation by As(III)
have been determined. The perturbed Trp shows a large reduction in the overall decay
lifetime compared with unperturbed Trp residue, exhibiting a high selectively for
the Tx sublevel. This selectivity suggests that the As atom lies in the xz plane of
the principal magnetic axis system of Trp, but not directly along the z (out-of-plane)
axis. The accessibility of this enzyme binding site to the arsenical is decreased
upon forming a ternary complex of methylase with sinefungin and a DNA oligomer, d[GCGAA(BrU)(BrU)CGC],
containing two 5-bromouracil (BrU) bases in place of thymine within the hexadeoxynucleotide
recognition sequence. This result indicates that the arsenical binding site in methylase
which produces the Trp heavy-atom effect is protected from this ligand by ternary
complex formation or the enzyme undergoes a conformation change, removing the Cys
from the Trp site. This protection is also observed in fluorescence quenching experiments.(ABSTRACT
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