Determination of binding affinity of metal cofactor to the active site of methionine aminopeptidase based on quantitation of functional enzyme.

Determination of metal affinity to the active site of metalloenzymes constitutes an integral part in the understanding of enzyme catalysis and regulation. Nonlinear curve fitting of metal titration curves using the multiple independent binding sites (MIBS) model was adapted to determine K(D) values based on functional enzyme concentrations. This approach provides a more accurate evaluation of K(D) compared with existing methods that are based on total protein concentrations. We applied this concept to methionine aminopeptidase from Mycobacterium tuberculosis and showed that it is a monometalated enzyme with a K(D) of 0.13 microM for Co(2+).