Nitrogen-15 nuclear magnetic resonance of arsanilazotyrosine-248 carboxypeptidase A and its complex with β-phenylpropionate. Structure and dynamics in solution

Abstract

Nitrogen-15 nuclear magnetic resonance has been used to study the structure of arsanilazocarboxypeptidase A and its complex with the inhibitor β-phenylpropionate. Derivatives selectively enriched with ^(15)N were prepared to facilitate observation of the ^(15)N resonances. The results are consistent with the conclusions reached previously from absorption spectroscopic studies and, in addition, provide new information regarding the properties of the azoenzyme and its inhibitor complex. Direct evidence has been obtained for formation of an intramolecular complex between the catalytically essential zinc ion and azoTyr-248, and it has been possible to estimate the degree of complexation. Moreover, the zinc complex involves the distal (Nβ) nitrogen of the azo linkage, whereas a model compound, tetrazolyl-N-acetyltyrosine, complexes to zinc through the proximal (Nɑ) nitrogen. The ^(15)N NMR spectra give specific information regarding the intramolecular hydrogen bonding in the azoenzyme. The free azophenol form of the azoenzyme, like that of the model compound arsanilazo-N-acetyltyrosine, exists predominantly with the tyrosine phenolic proton intramolecularly hydrogen bonded to Nβ of the azo linkage to form a six-membered ring structure. A similar hydrogen bond is also present in the apoazoenzyme and in the azoenzyme-(Gly + L-Tyr) complex, but not in the complex between the azoenzyme and β-phenylpropionate. In the latter complex, there appears to be a new and strong hydrogen bond between the phenolic proton of Tyr-248 and the carboxylate group of enzyme- bound β-phenylpropionate. Thus, azoenzyme-bound β-phenylpropionate, but not azoenzyme-bound Gly + L-Tyr, is apparently able to compete effectively with, and displace, the azo nitrogen as the hydrogen-bond acceptor of the phenolic proton of Tyr-248.

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