Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

2011 ◽  
Vol 441 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Zi-Ren Zhou ◽  
Yu-Hang Zhang ◽  
Shuai Liu ◽  
Ai-Xin Song ◽  
Hong-Yu Hu
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Dependent Manner ◽  
Deubiquitinating Enzymes ◽  
Chain Flexibility ◽  
Isopeptide Bond ◽  
Short Loop ◽  
The One ◽  
Isopeptidase Activity ◽  
Hydrolysis Of

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.

scholarly journals A comparison of the active site of maltase-glucoamylase from the brush border of rabbit small intestine and kidney by chemical modification studies

1991 ◽  
Vol 274 (2) ◽  
pp. 349-354 ◽  
Author(s):  
B Pereira ◽  
S Sivakami
Keyword(s):  
Chemical Modification ◽  
Brush Border ◽  
Active Site ◽  
Brush Border Membrane ◽  
Kidney Enzyme ◽  
Rabbit Intestine ◽  
Rabbit Small Intestine ◽  
Intestinal Enzyme ◽  
The One ◽  
Hydrolysis Of

The neutral maltase-glucoamylase complex has been purified to homogeneity from the brush-border membrane of rabbit intestine and kidney. Chemical modification of the amino acid side chains was carried out on the purified enzymes. Studies on the kidney enzyme revealed that tryptophan, histidine and cysteine were essential for both maltase and glucoamylase activities, whereas tryptophan, histidine and lysine were essential for the maltase and glucoamylase activities of the intestinal enzyme. Though there was no difference in the amino acids essential for the hydrolysis of maltose and starch by any one enzyme, starch hydrolysis seems to require two histidine residues instead of the one which is required for maltose hydrolysis. This appears to be true for both the intestinal and kidney enzymes.

scholarly journals Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

2005 ◽  
Vol 49 (8) ◽  
pp. 3421-3427 ◽  
Author(s):  
Fahd K. Majiduddin ◽  
Timothy Palzkill
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Class A ◽  
Study Support ◽  
Functional Mutants ◽  
Hydrolysis Of ◽  
Traditional Class

ABSTRACT Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

scholarly journals Processivity, Synergism, and Substrate Specificity of Thermobifida fusca Cel6B

2009 ◽  
Vol 75 (21) ◽  
pp. 6655-6661 ◽  
Author(s):  
Thu V. Vuong ◽  
David B. Wilson
Keyword(s):  
Substrate Specificity ◽  
Microcrystalline Cellulose ◽  
Filter Paper ◽  
Active Site ◽  
Carboxymethyl Cellulose ◽  
Inverse Relationship ◽  
Biomass Conversion ◽  
Thermobifida Fusca ◽  
Effective Strategy ◽  
Hydrolysis Of

ABSTRACT A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.

scholarly journals Substrate specificity and kinetic properties of α-galactosidases from Vicia faba

1969 ◽  
Vol 115 (1) ◽  
pp. 47-54 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Catalytic Activity ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Vicia Faba ◽  
Active Site ◽  
Effect Of Temperature ◽  
Kinetic Properties ◽  
Photo Oxidation ◽  
Hydrolysis Of ◽  
Enzyme I

1. The hydrolysis of a variety of galactosides and other glycosides by α-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl α-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on Km and Vmax. values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

A crystallographic study of the oxidation of lysozyme by iodine

1967 ◽  
Vol 167 (1009) ◽  
pp. 435-438 ◽  
Keyword(s):  
Active Site ◽  
The Other ◽  
Enzymic Hydrolysis ◽  
Substrate Modification ◽  
Crystallographic Study ◽  
Competitive Inhibitors ◽  
Plausible Hypothesis ◽  
The One ◽  
Hydrolysis Of ◽  
Tryptophanyl Residue

The crystallographic examinations of the lysozyme molecule, and of the complexes of lysozyme with competitive inhibitors, have resulted in the identification of the active site and in a plausible hypothesis for its activity as described in the previous papers at this meeting (Blake, Mair, North, Phillips & Sarma, p. 365; Blake, Johnson, Mair, North, Phillips & Sarma, p. 378). The active site includes three tryptophanyl residues (62, 63 and 108) which appear to be involved in the binding of the substrate. Modification of some or all of these residues may provide further evidence of their function in the activity of the enzyme (see figure 19 a of Blake et al ., p. 384). Hartdegen & Rupley (1964) showed that the action of small amounts of triiodide produced two modified lysozymes. One product, containing iodine, was enzymically active, while the other which contained no iodine was inactive. Enzymic hydrolysis of the latter product revealed that it had one less tryptophanyl residue than the native enzyme. Hartdegen & Rupley were not able, however, to show which of the six tryptophanyl residues in the lysozyme molecule was the one uniquely reactive to iodine, but they were able to show that it was part of, or near, the active site.

scholarly journals Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase

1987 ◽  
Vol 246 (2) ◽  
pp. 537-541 ◽  
Author(s):  
A R Plant ◽  
R M Clemens ◽  
H W Morgan ◽  
R M Daniel
Keyword(s):  
Kinetic Model ◽  
Substrate Specificity ◽  
Chain Length ◽  
Carboxy Group ◽  
Active Site ◽  
Product Formation ◽  
Inactivation Kinetics ◽  
Short Chain Length ◽  
Ph Dependent ◽  
Hydrolysis Of

Thermoanaerobium Tok6-B1 pullulanase (EC 3.2.1.41) was active on alpha 1-6-glucosidic linkages of pullulan, amylopectin and glycogen and the alpha 1-4 linkages of amylose, amylopectin and glycogen but not of pullulan. Hydrolysis of short-chain-length malto-oligosaccharides (seven or fewer glucose residues) yielded maltose as product. Pullulan hydrolysis was pH-dependent and a plot of log(V/Km) versus pH implied a carboxy group with pKa 4.3 at the active site. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide (EDAC) confirmed this view, and analysis of the order of reaction and inactivation kinetics suggested the presence of a single carboxy group at a catalytic centre of the active site. EDAC-mediated inhibition of pullulan alpha 1-6-bond hydrolysis was relieved by amylose or pullulan. Similarly both pullulan and amylose protected the activity directed at alpha 1-4 bonds of amylose from EDAC inhibition. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which both substrates were hydrolysed at the same active site.

The Effects of Amines on the Esterase Activities of Thrombin and Thrombokinase and on Several Clotting Tests

1974 ◽  
Vol 31 (02) ◽  
pp. 309-318
Author(s):  
Phyllis S Roberts ◽  
Raphael M Ottenbrite ◽  
Patricia B Fleming ◽  
James Wigand
Keyword(s):  
Choline Chloride ◽  
Polymerization Process ◽  
Ammonium Bromide ◽  
Bovine Thrombin ◽  
One Stage ◽  
Human Proteins ◽  
Rate Of Hydrolysis ◽  
The One ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

Summary1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis of this ester by either human or bovine thrombin. Only when 1.0 M or more choline chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly inhibited. Evidence is presented that shows that these effects are due to the quaternary amine group.2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and -n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction. In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on these reactions, however, do not follow any regular order. The tetraethyl compound is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction. The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME and the propyl compound of the thrombin-BAME reactions.3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified one-stage prothrombin test. In all cases the inhibition increases regularly as the size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025 M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage tests by the quaternary amines is not due to any effect of the com pounds on the polymerization process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary amines.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

scholarly journals Oligoribonuclease is the primary degradative enzyme for pGpG inPseudomonas aeruginosathat is required for cyclic-di-GMP turnover

2015 ◽  
Vol 112 (36) ◽  
pp. E5048-E5057 ◽  
Author(s):  
Mona W. Orr ◽  
Gregory P. Donaldson ◽  
Geoffrey B. Severin ◽  
Jingxin Wang ◽  
Herman O. Sintim ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Half Life ◽  
Biofilm Formation ◽  
Degradation Pathway ◽  
Dependent Manner ◽  
Capillary Action ◽  
Diguanylate Cyclases ◽  
Active Site Mutants ◽  
Degradative Enzyme

The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A fromPseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆ornstrains ofP. aeruginosaPA14 for pGpG stability. The lysates from ∆ornshowed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆ornstrain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP–regulatedpelpromoter. Additionally, the c-di-GMP–governed auto-aggregation and biofilm phenotypes were elevated in the ∆ornstrain in apel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆ornstrain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.

scholarly journals Erratum to: “Self-assembling catalytic systems based on new amphiphile containing purine fragment, exhibiting substrate specificity in hydrolysis of phosphorus acids esters”

2017 ◽  
Vol 87 (7) ◽  
pp. 1649-1649
Author(s):  
D. A. Samarkina ◽  
D. R. Gabdrakhmanov ◽  
V. E. Semenov ◽  
F. G. Valeeva ◽  
L. M. Gubaidullina ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Catalytic Systems ◽  
Self Assembling ◽  
Hydrolysis Of
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