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Functional requirement for human pitrilysin metallopeptidase 1 arginine 183, mutated in amyloidogenic neuropathy.
Protein Science (2018)
  • Jillian E. Smith-Carpenter
  • Benjamin J. Alper
Here we report the enzymologic characterization of recombinant human pitrilysin metallopeptidase 1 (Pitrm1) and derivative mutants including the arginine-to-glutamine substitution mutant Pitrm1 R183Q, which has been implicated in inherited amyloidogenic neuropathy. Recombinant Pitrm1 R183Q was readily expressed in and purified from Escherichia coli, but was less active than the recombinant wild-type enzyme against recombinant amyloid beta-peptide (Aβ 1-40). A novel fluorogenic substrate derived from the reported Aβ 1-40 core peptide cleavage sequence, Mca-KLVFFAEDK-(Dnp)-OH, was synthesized and applied to real-time kinetic study of Pitrm1 and derivative mutants including Pitrm1 R183Q. The Pitrm1 R183Q mutant exhibited significantly decreased rate of fluorogenic peptide hydrolysis, yet retained similar binding affinity by comparison with the wild-type enzyme. Targeted mutagenic analysis revealed a functional requirement for uncharged or electropositive residues in place of Pitrm1 R183. Residue R183 is positioned within an N-terminal strand-loop-strand motif that is conserved among M16C, but not M16A or M16B family metallopeptidases. Truncation analysis revealed that this strand-loop-strand motif inclusive of residue R183 is essential Pitrm1 function. A requirement for charged residues within 4.5 Å of residue R183 was demonstrated, and Pitrm1 R183Q was found to exhibit increased sensitivity to heat inactivation. Our findings indicate that charge sharing in the vicinity of Pitrm1 R183 is critical to enzyme activity, providing potential insight into a molecular basis of Pitrm1 dysfunction.
  • M16C peptidase,
  • amyloid beta-peptide (Aβ),
  • amyloidosis,
  • matrix metallopeptidase,
  • neuropathy,
  • presequence peptidase
Publication Date
Spring April, 2018
Citation Information
Jillian E. Smith-Carpenter and Benjamin J. Alper. "Functional requirement for human pitrilysin metallopeptidase 1 arginine 183, mutated in amyloidogenic neuropathy." Protein Science Vol. 27 Iss. 4 (2018) p. 861 - 873
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