Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embyogenesis and development

Journal of Biological Chemistry 2006, 281, 10737–10744

Giordano F.Z. da Silva,1 Rae L. Reuille,2 Li-June Ming,1 and Brian T. Livingston2

Departments of Chemistry1 and Biology2, University of South Florida, Tampa, FL 33620

Blastula Protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis which has been assigned to the astacin family of Zn-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like (TLL) genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 1 (BMP-1). Astacin, a crustacean digestive enzyme, has been proposed to carry out hydrolysis via a metal-centered mechanism that involves a metal-coordinated “tyrosine switch”. It has not been determined if the more structurally complex members of this family involved in eukaryotic development share this mechanism. The recombinant BP10 has been overexpressed in Escherichia coli, its metalloenzyme nature confirmed, and its catalytic properties characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native Zn-containing form and copper derivative. The copper derivative of BP10 shows a remarkable 960% rate acceleration toward the hydrolysis of the synthetic substrate N-benzoyl-arginine-p-nitroanilide when compared to the Zn form. The enzyme also shows calcium-dependent activation. These are the first thorough mechanistic studies reported on BP10 as a representative of the more structurally complex members of astacin-type enzymes in deuterostomes which can add supporting data to corroborate the metal-centered mechanism proposed for astacin and the role of the coordinated Tyr. We have demonstrated the first mechanistic study of a tolloid related metalloenzyme involved in sea urchin embryogenesis.