scholarly journals Flexibility in substrate recognition by thimet oligopeptidase as revealed by denaturation studies

2005 ◽  
Vol 388 (1) ◽  
pp. 255-261 ◽  
Author(s):  
Jeffrey A. SIGMAN ◽  
Tasneem H. PATWA ◽  
Ana V. TABLANTE ◽  
Calleen D. JOSEPH ◽  
Marc J. GLUCKSMAN ◽  
...  
Keyword(s):  
Tertiary Structure ◽  
Catalytic Domain ◽  
Fluorescence Emission ◽  
Complete Loss ◽  
Second Step ◽  
Binding Motif ◽  
Substrate Selectivity ◽  
Collisional Quenching ◽  
Emission Anisotropy ◽  
Thimet Oligopeptidase

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.

scholarly journals The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain

2010 ◽  
Vol 21 (18) ◽  
pp. 3232-3246 ◽  
Author(s):  
Yi Ting Zhou ◽  
Li Li Chew ◽  
Sheng-cai Lin ◽  
Boon Chuan Low
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Intramolecular Interaction ◽  
Complete Loss ◽  
Protein Domain ◽  
Binding Motif ◽  
Gtpase Activating Protein ◽  
New Paradigm ◽  
Cell Rounding ◽  
In Cis

The BNIP-2 and Cdc42GAP homology (BCH) domain is a novel regulator for Rho GTPases, but its impact on p50-Rho GTPase-activating protein (p50RhoGAP or Cdc42GAP) in cells remains elusive. Here we show that deletion of the BCH domain from p50RhoGAP enhanced its GAP activity and caused drastic cell rounding. Introducing constitutively active RhoA or inactivating GAP domain blocked such effect, whereas replacing the BCH domain with endosome-targeting SNX3 excluded requirement of endosomal localization in regulating the GAP activity. Substitution with homologous BCH domain from Schizosaccharomyces pombe, which does not bind mammalian RhoA, also led to complete loss of suppression. Interestingly, the p50RhoGAP BCH domain only targeted RhoA, but not Cdc42 or Rac1, and it was unable to distinguish between GDP and the GTP-bound form of RhoA. Further mutagenesis revealed a RhoA-binding motif (residues 85-120), which when deleted, significantly reduced BCH inhibition on GAP-mediated cell rounding, whereas its full suppression also required an intramolecular interaction motif (residues 169-197). Therefore, BCH domain serves as a local modulator in cis to sequester RhoA from inactivation by the adjacent GAP domain, adding to a new paradigm for regulating p50RhoGAP signaling.

scholarly journals Insights into the Maturation of Pernisine, a Subtilisin-Like Protease from the Hyperthermophilic Archaeon Aeropyrum pernix

2020 ◽  
Vol 86 (17) ◽  
Author(s):  
Miha Bahun ◽  
Marko Šnajder ◽  
Dušan Turk ◽  
Nataša Poklar Ulrih
Keyword(s):  
High Temperature ◽  
High Temperatures ◽  
Catalytic Domain ◽  
Industrial Applications ◽  
Binding Motif ◽  
Content Type ◽  
Hyperthermophilic Archaeon ◽  
Aeropyrum Pernix ◽  
Autocatalytic Process ◽  
Exceptional Stability

ABSTRACT Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix. Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism. IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.

Kinetic and structural analysis of the ultrasensitive behaviour of cyanobacterial ADP-glucose pyrophosphorylase

2000 ◽  
Vol 350 (1) ◽  
pp. 139-147 ◽  
Author(s):  
Diego F. GÓMEZ CASATI ◽  
Miguel A. AON ◽  
Alberto A. IGLESIAS
Keyword(s):  
Conformational Changes ◽  
Tertiary Structure ◽  
Fluorescence Emission ◽  
Maximal Activity ◽  
Size Exclusion ◽  
Exclusion Chromatography ◽  
Adp Glucose Pyrophosphorylase ◽  
Kinetics Of ◽  
Allosteric Regulators

The kinetic and (supra)molecular properties of the ultrasensitive behaviour of ADP-glucose pyrophosphorylase (AGPase) from Anabaena PCC 7120 (a cyanobacterium) were exhaustively studied. The response of the enzyme toward the allosteric activator 3-phosphoglycerate (3PGA) occurs with ultrasensitivity as a consequence of the cross-talk with the inhibitor Pi. Molecular ‘crowding’renders AGPase more sensitive to the interplay between the allosteric regulators and, consequently, enhances the ultrasensitive response. In crowded media, and when orthophosphate is present, the activation kinetics of the enzyme with 3PGA proceed with increased co-operativity and reduced affinity toward the activator. Under conditions of ultrasensitivity, the enzyme's maximal activation takes place in a narrow range of 3PGA concentrations. Moreover, saturation kinetics of the enzyme with respect to its substrates, glucose 1-phosphate and ATP, were different at low or high 3PGA levels in crowded media. Only under the latter conditions did AGPase exhibit discrimination between low or high levels of the activator, which increased the affinity toward the substrates and the maximal activity reached by the enzyme. Studies of fluorescence emission of tryptophan residues, fourth-derivative spectroscopy and size-exclusion chromatography indicated that the ultrasensitive behaviour is correlated with intramolecular conformational changes induced in the tertiary structure of the homotetrameric enzyme. The results suggest a physiological relevance of the ultrasensitive response of AGPase in vivo, since the enzyme could be subtly sensing changes in the levels of allosteric regulators and substrates, and thus determining the flux of metabolites toward synthesis of storage polysaccharides.

scholarly journals The structure of the colorectal cancer-associated enzyme GalNAc-T12 reveals how nonconserved residues dictate its function

2019 ◽  
Vol 116 (41) ◽  
pp. 20404-20410 ◽  
Author(s):  
Amy J. Fernandez ◽  
Earnest James Paul Daniel ◽  
Sai Pooja Mahajan ◽  
Jeffrey J. Gray ◽  
Thomas A. Gerken ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Molecular Basis ◽  
Catalytic Domain ◽  
Binding Pocket ◽  
Substrate Selectivity ◽  
Peptide Substrate ◽  
X Ray ◽  
Biochemical Studies ◽  
Cancer Mutations ◽  
Insight Into

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.

Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity

2016 ◽  
Vol 180 (6) ◽  
pp. 1056-1075 ◽  
Author(s):  
Edgar Omar Mendoza-Llerenas ◽  
David Javier Pérez ◽  
Zeferino Gómez-Sandoval ◽  
Pilar Escalante-Minakata ◽  
Vrani Ibarra-Junquera ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Catalytic Domain ◽  
Substrate Selectivity

scholarly journals gdt1, a New Signal Transduction Component for Negative Regulation of the Growth–Differentiation Transition in Dictyostelium discoideum

2000 ◽  
Vol 11 (5) ◽  
pp. 1631-1643 ◽  
Author(s):  
Changjiang Zeng ◽  
Christophe Anjard ◽  
Karsten Riemann ◽  
Angelika Konzok ◽  
Wolfgang Nellen
Keyword(s):  
Catalytic Domain ◽  
Complete Loss ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Developmental Cycle ◽  
Wild Type ◽  
C Terminus ◽  
Parallel Pathway ◽  
Remi Mutagenesis ◽  
Different Parts

Discoidin I expression was used as a marker to screen for mutants affected in the growth–differentiation transition (GDT) ofDictyostelium. By REMI mutagenesis we have isolated mutant 2-9, an overexpressor of discoidin I. It displays normal morphogenesis but shows premature entry into the developmental cycle. The disrupted gene was denominated gdt1. The mutant phenotype was reconstructed by disruptions in different parts of the gene, suggesting that all had a complete loss of function.gdt1 was expressed in growing cells; the levels of protein and mRNA appear to increase with cell density and rapidly decrease with the onset of development. gdt1 encodes a 175-kDa protein with four putative transmembrane domains. In the C terminus, the derived amino acid sequence displays some similarity to the catalytic domain of protein kinases. Mixing experiments demonstrate that the gdt1 −phenotype is cell autonomous. Prestarvation factor is secreted at wild-type levels. The response to folate, a negative regulator of discoidin expression, was not impaired in gdt1 mutants. Cells that lack the G protein α2 display a loss of discoidin expression and do not aggregate.gdt1 −/Gα2 −double mutants show no aggregation but strong discoidin expression. This suggests that gdt1 is a negative regulator of the GDT downstream of or in a parallel pathway to Gα2.

Intermediate studies on refolding of arginine kinase denatured by guanidine hydrochloride

2005 ◽  
Vol 83 (2) ◽  
pp. 109-114 ◽  
Author(s):  
Hong-Min Tang ◽  
Hong Yu
Keyword(s):  
Fluorescence Intensity ◽  
Tertiary Structure ◽  
Structural Characteristics ◽  
Molten Globule ◽  
Guanidine Hydrochloride ◽  
Arginine Kinase ◽  
Fluorescence Emission ◽  
Tryptophan Fluorescence ◽  
Blue Shift ◽  
Molten Globule State

The refolding course and intermediate of guanidine hydrochloride (GuHCl)-denatured arginine kinase (AK) were studied in terms of enzymatic activity, intrinsic fluorescence, 1-anilino-8-naphthalenesulfonte (ANS) fluorescence, and far-UV circular dichroism (CD). During AK refolding, the fluorescence intensity increased with a significantly blue shift of the emission maximum. The molar ellipticity of CD increased to close to that of native AK, as compared with the fully unfolded AK. In the AK refolding process, 2 refolding intermediates were observed at the concentration ranges of 0.8–1.0 mol/L and 0.3–0.5 mol GuHCl/L. The peak position of the fluorescence emission and the secondary structure of these conformation states remained roughly unchanged. The tryptophan fluorescence intensity increased a little. However, the ANS fluorescence intensity significantly increased, as compared with both the native and the fully unfolded states. The first refolding intermediate at the range of 0.8–1.0 mol GuHCl/L concentration represented a typical "pre-molten globule state structure" with inactivity. The second one, at the range of 0.3–0.5 mol GuHCl/L concentration, shared many structural characteristics of native AK, including its secondary and tertiary structure, and regained its catalytic function, although its activity was lower than that of native AK. The present results suggest that during the refolding of GuHCl-denatured AK there are at least 2 refolding intermediates; as well, the results provide direct evidence for the hierarchical mechanism of protein folding.Key words: arginine kinase, guanidine-denatured, refolding, intermediate, molten globule state.

scholarly journals p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin-dependent kinases.

1997 ◽  
Vol 17 (8) ◽  
pp. 4338-4345 ◽  
Author(s):  
P Saha ◽  
Q Eichbaum ◽  
E D Silberman ◽  
B J Mayer ◽  
A Dutta
Keyword(s):  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Catalytic Domain ◽  
Cyclin E ◽  
Binding Motif ◽  
Cdk Inhibitors ◽  
Tgf Beta ◽  
Myc Oncogene

Cdc25A, a phosphatase essential for G1-S transition, associates with, dephosphorylates, and activates the cell cycle kinase cyclin E-cdk2. p21CIP1 and p27 are cyclin-dependent kinase (cdk) inhibitors induced by growth-suppressive signals such as p53 and transforming growth factor beta (TGF-beta). We have identified a cyclin binding motif near the N terminus of Cdc25A that is similar to the cyclin binding Cy (or RR LFG) motif of the p21CIP1 family of cdk inhibitors and separate from the catalytic domain. Mutations in this motif disrupt the association of Cdc25A with cyclin E- or cyclin A-cdk2 in vitro and in vivo and selectively interfere with the dephosphorylation of cyclin E-cdk2. A peptide based on the Cy motif of p21 competitively disrupts the association of Cdc25A with cyclin-cdks and inhibits the dephosphorylation of the kinase. p21 inhibits Cdc25A-cyclin-cdk2 association and the dephosphorylation of cdk2. Conversely, Cdc25A, which is itself an oncogene up-regulated by the Myc oncogene, associates with cyclin-cdk and protects it from inhibition by p21. Cdc25A also protects DNA replication in Xenopus egg extracts from inhibition by p21. These results describe a mechanism by which the Myc- or Cdc25A-induced oncogenic and p53- or TGF-beta-induced growth-suppressive pathways counterbalance each other by competing for cyclin-cdks.

scholarly journals Depolarization of the intrinsic and extrinsic fluorescence of pepsinogen and pepsin

1970 ◽  
Vol 116 (3) ◽  
pp. 341-348 ◽  
Author(s):  
F. W. J. Teale ◽  
R. A. Badley
Keyword(s):  
Relaxation Times ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Molecule ◽  
Rotational Relaxation ◽  
Collisional Quenching ◽  
Shape Factors ◽  
Emission Anisotropy ◽  
Specific Orientation ◽  
Tryptophan Emission

1. The effects on the intrinsic tryptophan emission anisotropy of pepsin and pepsinogen solutions produced by (a) changes in temperature, (b) increases in viscosity with added glycerol at constant temperature and (c) decreases in lifetime through collisional quenching by potassium iodide were measured at several excitation wavelengths. The rotational-relaxation times calculated from results provided by method (b) approximate to the theoretical values for the two proteins, on taking hydration and shape factors into account, on the basis of random orientation of the tryptophan groups within the macromolecules. Differences between the results provided by methods (b) and (c) are attributable to inter-tryptophan resonance-energy-transfer depolarization, and the anomalous values recorded in method (a) can be attributed to the temperature-dependence of the limiting anisotropies. 2. Two different monomeric conjugates of pepsin, each containing one extrinsic fluorescent group per macromolecule, gave widely different relaxation times. This difference may arise from a specific orientation of the emission dipole in the enzyme. In active-site-labelled pepsin (1-dimethylaminonaphthalene-5-sulphonylphenylalanine–pepsin) this orientation would be approximately parallel to the symmetry axis of the equivalent ellipsoid, whereas in the other conjugate (1-dimethylaminonaphthalene-5-sulphonyl-pepsin) the orientation may be roughly normal to this direction, or some independent rotation of parts of the protein molecule is possible.

Prediction of the Tertiary Structure of α-Glycosidase from Aspergillus niger by Homology Modeling

2011 ◽  
Vol 399-401 ◽  
pp. 2160-2163
Author(s):  
Fa Xiang Wang ◽  
Qin Yun Wang ◽  
Yong Le Liu ◽  
Jian Yu
Keyword(s):  
Homology Modeling ◽  
Tertiary Structure ◽  
Catalytic Domain ◽  
Molecular Model ◽  
Critical Role ◽  
Three Dimensional ◽  
Primary Metabolism ◽  
Structural Domains ◽  
Conserved Residues ◽  
Substrate Binding Sites

α-Glucosidases play critical role both in primary metabolism and in glycoconjugate biosynthesis and processing. In this paper, the reasonable three-dimensional molecular model of AglA was generated by homology modeling. This modeled protein is divided into five major structural domains, and the catalytic domain is classical (β/α) 8 barrel with the active site pocket positioned at its C-terminal side. With analyses of conserved residues and overlay of homology structures, the residues Tyr 662, Tyr527, Glu521, His238 and Tyr235 was predicted as the main substrate binding sites, and residues Asp490, Glu493 and Asp660 were deduced to be the acid/base catalytic residues.

Sign in / Sign up

Export Citation Format

Share Document