Michael N. Harris and Li-June Ming*
Department of Chemistry and Institute for Biomolecular Science
University of South Florida
Tampa, FL 33620-5250
Phosphate shows a noncompetitive inhibition toward a Streptomyces aminopeptidase (sAP) between pH 5.85 (Ki = 0.48 mM) and 9.0 (110 mM), with a pKa of 7.05 likely due to ionization of H2PO4–. This noncompetitive inhibition pattern indicates that phosphate binding to sAP in solution is different from that in the crystal structure, where phosphate is bound to the active site Zn(II) ions. Fluoride uncompetitively inhibits sAP from pH 5.5 (Ki = 3.72 mM) to 9.0 (43.6 mM), with a pKa of ~6.2 likely due to a coordinated water. The different inhibition natures and pKa values indicate that the two inhibitors bind at different locations.
Crystallographic studies of transition state analogs with blAP and aAP suggest that the zinc ions function in the binding and polarization of the substrate. The phosphate inhibition study clearly shows that phosphate and the substrate bind to sAP at different locations. Therefore, the binding of phosphate to sAP in solution cannot be the same as that proposed from the crystal structure. This conclusion is supported by our preliminary 31P NMR studies of phosphate binding to CoZn-sAP, which do not suggest direct phosphate-metal interaction. A detailed investigation of phosphate binding to the Co2+ derivative of sAP by the use of NMR is in progress. Fluoride inhibition has provided insight into a zinc-bound coordinated water of sAP. The uncompetitive inhibition pattern of F– towards sAP is similar to that observed in aAP and can suggest possible mechanistic similarities between the two enzymes despite their low sequence homology (24% in aligned regions) and very different functional groups in the proximity of the active site zinc ions.
Acknowledgments: We are grateful for some measurements and data analyses conducted by Rebecca A. Konnerth, Andrew Kuo, Akshata A. Marballi, Michelle C. Ming who were in the Summer BioMed and Life Sciences Program for Gifted/Talented High School Students at the University of South Florida. We are also grateful for the preliminary results on phosphate inhibition obtained by Rosemary Keene. The supports provided by the University and the Summer Program are acknowledged.
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