Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies.
نویسندگان
چکیده
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.
منابع مشابه
Swapping the substrate specificities of the neuropeptidases neurolysin and thimet oligopeptidase.
Thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) are closely related zinc-dependent metallopeptidases that metabolize small bioactive peptides. They cleave many substrates at the same sites, but they recognize different positions on others, including neurotensin, a 13-residue peptide involved in modulation of dopaminergic circuits, pain perception, and thermoregulation. On the...
متن کاملHuman thimet oligopeptidase.
We have purified human thimet oligopeptidase to homogeneity from erythrocytes, and compared it with the enzyme from rat testis and chicken liver. An antiserum raised against rat thimet oligopeptidase also recognized the human and chicken enzymes, suggesting that the structure of the enzyme has been strongly conserved in evolution. Consistent with this, the properties of the human enzyme were ve...
متن کاملCrystal structure of human thimet oligopeptidase provides insight into substrate recognition, regulation, and localization.
Thimet oligopeptidase (TOP) is a zinc metallopeptidase that metabolizes a number of bioactive peptides and degrades peptides released by the proteasome, limiting antigenic presentation by MHC class I molecules. We present the crystal structure of human TOP at 2.0-A resolution. The active site is located at the base of a deep channel that runs the length of the elongated molecule, an overall fol...
متن کاملThimet oligopeptidase: similarity to 'soluble angiotensin II-binding protein' and some corrections to the published amino acid sequence of the rat testis enzyme.
The deduced amino acid sequence of pig liver soluble angiotensin II-binding protein [Sugiura, Hagiwara and Hirose (1992) J. Biol. Chem. 267, 18067-18072] is similar over most of its length to that reported for rat testis thimet oligopeptidase (EC 3.4.24.15) by Pierotti, Dong, Glucksman, Orlowski and Roberts [(1990) (Biochemistry 29, 10323-10329]. We have found that homogeneous rat testis thimet...
متن کاملExtracellular Thimet Oligopeptidase is Carried by Cell Membrane Microvesicles of Human Prostate Cancer Cells
Extracellular Thimet Oligopeptidase is Carried by Cell Membrane Microvesicles of Human Prostate Cancer Cells
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- The Biochemical journal
دوره 388 Pt 1 شماره
صفحات -
تاریخ انتشار 2005