Circular dichroism-inhibitor titrations of arsanilazotyrosine-248 carboxypeptidase A.
Coupling of carboxypeptidase with diazotized arsanilic acid specifically modifies a single tyrosyl residue. Yet, owing to the fact that the resultant azoTyr-248 can form an intramolecular chelate with zinc, two different circular dichroism probes result: azoTyr-248 itself and the azoTyr-248-Zn chelate. Both are environmentally sensitive and, characteristically, each can signal the same or different perturbations, as is apparent from circular dichroic spectra. This dual probe function greatly magnifies the scope of these chromophores in mapping the topography of the active center with respect to sites of interaction of inhibitors (or substrates). Titration of the azoenzyme with a series of synthetic, competitive inhibitors, e.g., L-benzylsuccinate, L-phenyllactate, and L-Phe, and with the pseudosubstrate, Gly-L-Tyr, in turn generates characteristic circular dichroic spectra. Their analysis yields a single binding constant for each of these agents, one molecule of each binding to the active center. Mixed inhibitions, as seen with beta-phenylpropionate and phenylacetate, resolved previously into competitive and noncompetitive components, are characterized by different spectral effects. Two molecules of these agents bind to the enzyme, consistent with both thermodynamic and enzymatic studies. The interactions leading to competitive and noncompetitive inhibition, respectively, can be recognized and assigned, based on the manner in which the extrema at 340 and 420 nm, reflecting azoTyr-248, and the negative 510-nm circular dichroism band, typical of its chelate with zinc, are affected and on the pH dependence of spectral and kinetic data. Certai4 noncompetitive inhibitors and modifiers induce yet other spectral features. Each probe is very sensitive to changes in its particular active center environment, though both can be relatively insensitive to inhibitors interacting at a distance from the active center.
D001151:Arsenic / Q000494:pharmacology; D001391:Azo Compounds / Q000494:pharmacology; D001665:Binding Sites; D002268:Carboxypeptidases / Q000037:antagonists & inhibitors; D002942:Circular Dichroism; D003201:Computers; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000032:analysis
D001391:Azo Compounds; D014443:Tyrosine; D002268:Carboxypeptidases; D001151:Arsenic