Conformations of Arsanilazotyrosine-248 Carboxypeptidase A α,β,γ . Comparison of Crystals and Solution

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Abstract
The spectra of the α, β, and γ forms of zinc monoarsanilazotyrosine-248 carboxypeptidase A are indistinguishable. At pH 8.2 their crystals are yellow, while their solutions are red, λ max 510 nm. Absorption and circular dichroism-pH titrations of the modified zinc and apoenzymes demonstrate that the absorption band at 510 nm is due to a complex between arsanilazotyrosine-248 and the active-site zinc atom. Two pK app values, 7.7 and 9.5, characterize the formation and dissociation of this arsanilazotyrosine-248·Zn complex. On titrations of the apoenzyme, the absorption band at 510 nm is completely absent at all pH values. Instead, there is a single pK app , 9.4, due to the ionization of the azophenol, λ max 485 nm. Substitution of other metals for zinc results in analogous intramolecular coordination complexes with absorption maxima and circular dichroism extrema characteristic of the particular metal. Similar data and conclusions have been derived from studies of heterocyclic azophenol·metal complexes. The present studies demonstrate that the conformation of the crystals of all generally available α, β, and γ forms of the arsanilazoenzyme differs from that of their solutions. The spectra of the modified x-ray crystals, however, differ from those of all other carboxypeptidase forms and crystal habits studied. The internal consistency of their data, their interpretation, and the conclusions of Lipscomb and coworkers [ Proc. Nat. Acad. Sci. USA (1972) 69, 2850-2854] are examined. Dissimilar chemical modification or conformation is thought to underlie these differences. The arsanilazotyrosine-248·zinc complex is a sensitive, dynamic probe of environmental conditions. Its response to changes in pH and physical state of the enzyme suggest different orientation of the arsanilazotyrosine-248 side chain in solution from that in the crystal. This finding calls for reexamination of the basis of the substrate-induced conformation change which has been thought to be critical to the mechanism, postulated on the basis of the x-ray structure analysis performed at pH 7.5.