Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of catalytic events.
The intensely chromophoric intramolecular coordination complex formed between arsanilazotyrosine-248 and the active site zinc atom of azocarboxypeptidase A (Johansen, J. T. & Vallee, B. L. (1971) Proc. Nat. Acad. Sci. USA 68, 2532-2535) is a spectrokinetic probe of catalytic events. The interconversion of the azoTyr-248-Zn complex and its constituents is measured by stopped-flow pH and temperature-jump methods. The rate of interconversion, 64,000 sec-1, is orders of magnitude faster than that of the catalytic step itself (about 0.01-100 sec-1). Rapidly turned over peptide and ester substrates disrupt the azoTyr-248-Zn complex before hydrolysis occurs. As a consequence, formation of azoTyr-248, substrate binding, and catalysis can all be monitored while catalysis is actually in progress. The results of these dynamic studies specify a course of catalytic events, different from those postulated based on x-ray structure analysis. If azoTyr-248 is displaced, the direction is opposite to the inward movement postulated on the basis of x-ray studies and is not unique to induction by substrates, since rapid changes in pH also result in analogous spectral changes. AzoTyr-248 carboxypeptidase has all the features which are essential for mechanistic studies: (1) It is enzymatically active; (2) the spectra of the metal complex differ characteristically from those of its constituents; (3) it responds dynamically to environmental factors; and (4) the response time of the probe itself is much more rapid than is required for the measurement of the catalytic step. These combined kinetic and spectral properties of the metal complex render it a powerful spectrokinetic probe to visualize and discern microscopic details of the catalytic process.
D001152:Arsenicals; D001391:Azo Compounds; D001665:Binding Sites; D002268:Carboxypeptidases; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D013053:Spectrophotometry; D013696:Temperature; D014443:Tyrosine / Q000031:analogs & derivatives*; D015032:Zinc / Q000032:analysis; D010132:p-Azobenzenearsonate
D001152:Arsenicals; D001391:Azo Compounds; C000229:tyrosine-4-azobenzenearsonate; D014443:Tyrosine; D010132:p-Azobenzenearsonate; D002268:Carboxypeptidases; D015032:Zinc