title: Substitution of methionine 435 with leucine, isoleucine, and serine in tumor necrosis factor alpha converting enzyme inactivates ectodomain shedding activity.
abstract: Tumor necrosis factor alpha (TNF-alpha) converting enzyme (TACE) is a zinc metalloprotease that has emerged as a general sheddase, which is responsible for ectodomain release of numerous membrane proteins, including the proinflammatory cytokine TNF-alpha, the leukocyte adhesin L-selectin and epidermal growth factor receptor ligand-transforming growth factor alpha (TGF-alpha), and related family members. Structurally, TACE belongs to a large clan of proteases, designated the metzincins, because TACE possesses a conserved methionine (Met435), frequently referred to as the met-turn residue, in its active site. A vital role of this residue in the function of TACE is supported by the fact that cells expressing the M435I TACE variant are defective in ectodomain shedding. However, the importance of Met435 in TACE appears to be uncertain, since another metzincin, matrix metalloprotease-2, has been found to be enzymatically fully active with either leucine or serine in place of its met-turn residue. We constructed TACE mutants with leucine or serine in place of Met435 to further examine the role of the met-turn residue in TACE-mediated ectodomain cleavage. Similar to the M435I TACE mutant, both the M435L and M435S constructs are defective in cleaving transmembrane TNF-alpha, TGF-alpha, and L-selectin. Comparative modeling and dynamic computation detected structural perturbations, which resulted in higher energy, in the catalytic zinc complexes of the Met435 TACE mutants compared with that in the wild-type enzyme. Thus, Met435 serves to maintain the stability of the catalytic center of TACE for the hydrolysis of peptide bonds in substrates.
authors.affiliation: University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, 683 Hoes Lane, Piscataway, NJ 08854, USA.
authors.forename: Liliana
authors.initials: L
authors.lastname: Pérez
authors.forename: John E
authors.initials: JE
authors.lastname: Kerrigan
authors.forename: Xiaojin
authors.initials: X
authors.lastname: Li
authors.forename: Huizhou
authors.initials: H
authors.lastname: Fan
mesh_terms: D051722:ADAM Proteins / Q000235:genetics / Q000378:metabolism*; D000072198:ADAM17 Protein; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D020134:Catalytic Domain; D006224:Cricetinae; D007532:Isoleucine / Q000235:genetics*; D007930:Leucine / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism*; D008715:Methionine / Q000235:genetics*; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012694:Serine / Q000235:genetics*
chemical_list: D000596:Amino Acids; D008565:Membrane Proteins; D007532:Isoleucine; D012694:Serine; D008715:Methionine; D051722:ADAM Proteins; D000072198:ADAM17 Protein; D007930:Leucine