Overexpression and mechanistic characterization of blastula protease 10, a metalloprotease involved in sea urchin embryogenesis and development.
Blastula protease 10 (BP10) is a metalloenzyme involved in sea urchin embryogenesis, which has been assigned to the astacin family of zinc-dependent endopeptidases. It shows greatest homology with the mammalian tolloid-like genes and contains conserved structural motifs consistent with astacin, tolloid, and bone morphogenetic protein 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 has been confirmed, and its catalytic properties have been characterized through kinetic studies. BP10 shows significant hydrolysis toward gelatin both in its native zinc-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 with the zinc 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.
da Silva
Giordano F Z
GF
Department of Chemistry, University of South Florida, Tampa, Florida 33620, USA.
Reuille
Rae L
RL
Ming
Li-June
LJ
Livingston
Brian T
BT
D000818:Animals; D001586:Benzoylarginine Nitroanilide / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D004625:Embryo, Nonmammalian / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D018093:Gelatinases / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008666:Metalloendopeptidases / Q000096:biosynthesis* / Q000737:chemistry*; D045726:Metalloproteases / Q000096:biosynthesis* / Q000737:chemistry; D008956:Models, Chemical; D011994:Recombinant Proteins / Q000737:chemistry; D012617:Sea Urchins; D013053:Spectrophotometry; D013379:Substrate Specificity; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D011994:Recombinant Proteins; D014443:Tyrosine; D003300:Copper; D001586:Benzoylarginine Nitroanilide; D045726:Metalloproteases; C100686:BP10 protein, Paracentrotus lividus; D018093:Gelatinases; D008666:Metalloendopeptidases; C071288:astacin; D015032:Zinc; D002118:Calcium