Alternative Activities of Metallobiomolecules

Ming, Li-June

Abstract

Recent research in our laboratory on the di-zinc aminopeptidase from Streptomyces griseus has laid a good foundation for the understanding of metal binding and the mechanism of this enzyme. We have also discovered that SgAP showed remarkable """"""""alternative activities"""""""" toward the hydrolyses of a phosphodiester bis(p-nitrophenyl) phosphate (BNPP) and a phosphonoester p-nitrophenylphosphonate (NPPP), reaching catalytic proficiency of 10 billion and 0.3 million, respectively, relative to auto-hydrolysis at neutral pH. Since phosphoesters and phosphonoesters are known to serve as transition-state inhibitors for proteases, the hydrolytic activities toward BNPP and NPPP are not supposed to occur, or at most not to take place effectively. Thus, it is important to address how this di-zinc enzyme performs this unexpected catalysis. This unique """"""""alternative enzymatic catalysis"""""""" provides a rare opportunity for the study of both peptide and phosphoester hydrolysis in a single enzyme system. Moreover, these """"""""alternative substrates"""""""" may serve as mechanistic probes for the study of the mechanism of this enzyme, which is otherwise not obtainable by the use of the """"""""regular"""""""" peptide substrates. To better understand this unusual alternative enzymatic catalysis and dinuclear hydrolysis, we propose the use of kinetic, thermodynamic, and nuclear magnetic resonance techniques in association with recombinant DNA techniques for the investigation of the mechanism of Streptomyces aminopeptidase toward regular peptide substrates and phosphoester """"""""alternative substrates"""""""". The similarity and difference between the normal and the alternative catalysis of the enzyme will be revealed and the secret code that engenders the unexpected catalysis of transition-state analogues will be uncovered by means of site-directed mutagenesis in association with the physical methods. As a result of the investigation, a comprehensive understanding of the structure and function of this dinuclear aminopeptidase can be achieved, which may provide a universal viewpoint about dinuclear hydrolysis in chemical and biological systems and may also provide further insight into substrate recognition and transition-state stabilization of enzymes.