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.
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