title: Catalytic Properties of Intramembrane Aspartyl Protease Substrate Hydrolysis Evaluated Using a FRET Peptide Cleavage Assay.
abstract: Chemical details of intramembrane proteolysis remain elusive despite its prevalence throughout biology. We developed a FRET peptide assay for the intramembrane aspartyl protease (IAP) from Methanoculleus marisnigri JR1 in combination with quantitative mass spectrometry cleavage site analysis. IAP can hydrolyze the angiotensinogen sequence, a substrate for the soluble aspartyl protease renin, at a predominant cut site, His-Thr. Turnover is slow (min(-1) × 10(-3)), affinity and Michaelis constant (Km) values are in the low micromolar range, and both catalytic rates and cleavage sites are the same in detergent as reconstituted into bicelles. Three well-established, IAP-directed inhibitors were directly confirmed as competitive, albeit with modest inhibitor constant (Ki) values. Partial deletion of the first transmembrane helix results in a biophysically similar but less active enzyme than full-length IAP, indicating a catalytic role. Our study demonstrates previously unappreciated similarities with soluble aspartyl proteases, provides new biochemical features of IAP and inhibitors, and offers tools to study other intramembrane protease family members in molecular detail.
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive Northwest, Atlanta, Georgia 30332-0400, United States.
authors.forename: Swe-Htet
authors.initials: SH
authors.lastname: Naing
authors.affiliation: School of Biology, Georgia Institute of Technology , 310 Ferst Drive Northwest, Atlanta, Georgia 30332-0230, United States.
authors.forename: Krishna M
authors.initials: KM
authors.lastname: Vukoti
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive Northwest, Atlanta, Georgia 30332-0400, United States.
authors.forename: Jason E
authors.initials: JE
authors.lastname: Drury
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive Northwest, Atlanta, Georgia 30332-0400, United States.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Johnson
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive Northwest, Atlanta, Georgia 30332-0400, United States.
authors.forename: Sibel
authors.initials: S
authors.lastname: Kalyoncu
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive Northwest, Atlanta, Georgia 30332-0400, United States.
authors.forename: Shannon E
authors.initials: SE
authors.lastname: Hill
authors.affiliation: School of Biology, Georgia Institute of Technology , 310 Ferst Drive Northwest, Atlanta, Georgia 30332-0230, United States.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Torres
authors.affiliation: School of Chemistry and Biochemistry, Georgia Institute of Technology , 901 Atlantic Drive Northwest, Atlanta, Georgia 30332-0400, United States.
authors.forename: Raquel L
authors.initials: RL
authors.lastname: Lieberman
mesh_terms: D000808:Angiotensinogen / Q000737:chemistry / Q000378:metabolism; D057055:Aspartic Acid Proteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D031541:Fluorescence Resonance Energy Transfer; D006868:Hydrolysis / Q000187:drug effects; D019638:Methanomicrobiaceae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D010455:Peptides; D000808:Angiotensinogen; D057055:Aspartic Acid Proteases