Direct verification of enzyme mechanisms requires knowledge of the structure of intermediates in catalysis. Metalloenzymes like carboxypeptidase A provide a unique opportunity in this regard because the metal is involved in catalysis and the structure of the metal binding site can be investigated in solution by X-ray Absorption Fine Structure, XAFS analysis. Experimentally, concurrent measurements of both structural and kinetic characteristics of such intermediates requires a lengthening of their very short lifetimes, e.g. by reducing their rates of formation and breakdown. This can be achieved by carrying out the reaction at low temperature and using rapid mixing devices. The ideal system is composed of several features all of which have the same fundamental objective i.e. detection and characterization of reaction intermediates without alteration of the reaction pathway. Of particular importance to these studies are the 1) means to detect structural changes, 2) ability to rapidly assay the enzyme at subzero temperatures and 3) crysolvent employed. Each of these features must be considered carefully for the enzyme under consideration. For CPD A three prominent features are its requirement for zinc for activity, its high reactivity toward N-terminal blocked peptides and depsipeptides and its solubility in salt solutions. Both of the former features can serve as a basis for creating suitable chromophoric probes. Substituting Co for Zn places a chromophoric atom at the active site of the enzyme which is sensitive to structural changes and the use of a fluorescent dansyl substrate blocking group can serve as the basis of a radiatioriless energy transfer assay of enzyme activity. These probes can aid in identifying substrates whose intermediates can be stabilized either by freeze quenching or equilibrium trapping at subzero temperatures, so that their structure can be determined by XAFS. In parallel experiments HPLC analysis will determine if the scissle bond is broken in the freeze quenched intermediate. XAFS allows structural comparisons of the metal binding site complexed with substrates or inhibitors for the solution and crystalline forms of both ZnCPD and CoCPD. The structure of the intermediates of CPD A will be compared to those obtained for other zinc proteases which have different zinc binding sites. The structure of these active site metal coordination complexes should be useful in assigning electronic transitions that occur in Co visible absorption, CD and MCD spectra to changes in the geometry of the complex as a whole or perhaps to movement of a single ligand.
Showing the most recent 10 out of 11 publications
