title: Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies.
abstract: Thimet oligopeptidase (TOP) is a soluble metalloendopeptidase belonging to a family of enzymes including neurolysin and neprilysin that utilize the HEXXH metal-binding motif. TOP is widely distributed among cell types and is able to cleave a number of structurally unrelated peptides. A recent focus of interest has been on structure-function relationships in substrate selectivity by TOP. The enzyme's structural fold comprises two domains that are linked at the bottom of a deep substrate-binding cleft via several flexible loop structures. In the present study, fluorescence spectroscopy has been used to probe structural changes in TOP induced by the chemical denaturant urea. Fluorescence emission, anisotropy and collisional quenching data support a two-step unfolding process for the enzyme in which complete loss of the tertiary structure occurs in the second step. Complete loss of activity and loss of catalytic Zn(II) from the active site, monitored by absorption changes of the metal chelator 4-(2-pyridylazo)-resorcinol, are also connected with the second step. In contrast, the first unfolding event, which is linked to changes in the non-catalytic domain, leads to a sharp increase in kcat towards a 9-residue substrate and a sharp decrease in kcat for a 5-residue substrate. Thus a conformational change in TOP has been directly correlated with a change in substrate selectivity. These results provide insight into how the enzyme can process the range of structurally unrelated peptides necessary for its many physiological roles.
authors.affiliation: Chemistry Department, Saint Mary's College of California, 1928 St. Mary's Road, Moraga, CA 94556, USA.
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Sigman
authors.forename: Tasneem H
authors.initials: TH
authors.lastname: Patwa
authors.forename: Ana V
authors.initials: AV
authors.lastname: Tablante
authors.forename: Calleen D
authors.initials: CD
authors.lastname: Joseph
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Glucksman
authors.forename: Adele J
authors.initials: AJ
authors.lastname: Wolfson
mesh_terms: D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D014508:Urea / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D014508:Urea; D008666:Metalloendopeptidases; C022510:thimet oligopeptidase; D015032:Zinc