scholarly journals Catalytic activity of bovine seminal ribonuclease is essential for its immunosuppressive and other biological activities

1995 ◽  
Vol 308 (2) ◽  
pp. 547-550 ◽  
Author(s):  
J S Kim ◽  
J Soucek ◽  
J Matousek ◽  
R T Raines
Keyword(s):  
Catalytic Activity ◽  
Quaternary Structure ◽  
Biological Activities ◽  
Biological Properties ◽  
Rnase A ◽  
Active Site Residue ◽  
Wild Type ◽  
Immunosuppressive Activity ◽  
Wild Type Enzyme ◽  
Bovine Seminal Ribonuclease

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with special biological properties, including potent immunosuppressive activity. A mutant BS-RNase was created in which His-119, the active-site residue that acts as a general acid during catalysis, was changed to an aspartic acid. H119D BS-RNase formed a dimer with quaternary structure similar to that of the wild-type enzyme but with values of kcat. and kcat./Km for the cleavage of UpA [uridylyl(3′-->5′)adenosine] that were 4 x 10(3)-fold lower. The mutant protein also demonstrated dramatically decreased immunosuppressive, anti-tumour, aspermatogenic, and embryotoxic activities. The catalytic activity of BS-RNase is therefore necessary for its special biological properties.

scholarly journals Aβ(M1–40) and Wild-Type Aβ40 Self-Assemble into Oligomers with Distinct Quaternary Structures

Molecules ◽  
2019 ◽  
Vol 24 (12) ◽  
pp. 2242 ◽  
Author(s):  
Jacob L. Bouchard ◽  
Taylor C. Davey ◽  
Todd M. Doran
Keyword(s):  
Self Assembly ◽  
Quaternary Structure ◽  
Biological Activities ◽  
Amyloid Β ◽  
Long Term Potentiation ◽  
Wild Type ◽  
Molecular Weights ◽  
Term Potentiation ◽  
Quaternary Structures

Amyloid-β oligomers (AβOs) self-assemble into polymorphic species with diverse biological activities that are implicated causally to Alzheimer’s disease (AD). Synaptotoxicity of AβO species is dependent on their quaternary structure, however, low-abundance and environmental sensitivity of AβOs in vivo have impeded a thorough assessment of structure–function relationships. We developed a simple biochemical assay to quantify the relative abundance and morphology of cross-linked AβOs. We compared oligomers derived from synthetic Aβ40 (wild-type (WT) Aβ40) and a recombinant source, called Aβ(M1–40). Both peptides assemble into oligomers with common sizes and morphology, however, the predominant quaternary structures of Aβ(M1–40) oligomeric states were more diverse in terms of dispersity and morphology. We identified self-assembly conditions that stabilize high-molecular weight oligomers of Aβ(M1–40) with apparent molecular weights greater than 36 kDa. Given that mixtures of AβOs derived from both peptides have been shown to be potent neurotoxins that disrupt long-term potentiation, we anticipate that the diverse quaternary structures reported for Aβ(M1–40) oligomers using the assays reported here will facilitate research efforts aimed at isolating and identifying common toxic species that contribute to synaptic dysfunction.

Role of catalytic and non-catalytic subsite residues in ribonuclease activity of human eosinophil-derived neurotoxin

2009 ◽  
Vol 390 (3) ◽  
Author(s):  
Deepa Sikriwal ◽  
Divya Seth ◽  
Janendra K. Batra
Keyword(s):  
Catalytic Activity ◽  
Biological Activities ◽  
Secretory Protein ◽  
Rnase A ◽  
Phosphate Binding ◽  
Human Eosinophil ◽  
Ribonucleolytic Activity ◽  
Phosphoryl Groups ◽  
Binding Subsites

Abstract Human eosinophil-derived neurotoxin (EDN), a secretory protein from eosinophils, is a member of the RNase A superfamily. The ribonucleolytic activity of EDN is central to its biological activities. EDN binds RNA in a cationic cleft, and the interaction between EDN and RNA substrate extends beyond the scissile bond. Based on its homology with RNase A, putative substrate binding subsites have been identified in EDN. The B1 and B2 subsites interact specifically with bases, whereas P0, P1, and P2 subsites interact with phosphoryl groups. In this study, we evaluated the role of putative residues of these subsites in the ribonucleolytic activity of EDN. We demonstrate that of the two base binding subsites, B1 is critical for the catalytic activity of EDN, as the substrate cleavage was dramatically reduced upon substitution of B1 subsite residues. Among the phosphate-binding subsites, P1 is the most crucial as mutations of its constituting residues totally abolished the catalytic activity of EDN. Mutation of P0 and P2 subsite residues only affected the catalytic activity on the homopolymer Poly(U). Our study demonstrates that P1 and B1 subsites of EDN are critical for its catalytic activity and that the other phosphate-binding subsites are involved in the activity on long homopolymeric substrates.

scholarly journals Selectivity of Fungal Sesquiterpene Synthases: Role of the Active Site's H-1α Loop in Catalysis

2010 ◽  
Vol 76 (23) ◽  
pp. 7723-7733 ◽  
Author(s):  
Fernando L�pez-Gallego ◽  
GraysonT. Wawrzyn ◽  
Claudia Schmidt-Dannert
Keyword(s):  
Marked Effect ◽  
Biological Activities ◽  
Site Directed Mutagenesis ◽  
Gas Chromatography Mass Spectrometry ◽  
Farnesyl Pyrophosphate ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Terpene Synthases ◽  
Product Profile

ABSTRACT Sesquiterpene synthases are responsible for the cyclization of farnesyl pyrophosphate into a myriad of structurally diverse compounds with various biological activities. We examine here the role of the conserved active site H-α1 loop in catalysis in three previously characterized fungal sesquiterpene synthases. The H-α1 loops of Cop3, Cop4, and Cop6 from Coprinus cinereus were altered by site-directed mutagenesis and the resultant product profiles were analyzed by gas chromatography-mass spectrometry and compared to the wild-type enzymes. In addition, we examine the effect of swapping the H-α1 loop from the promiscuous enzyme Cop4 with the more selective Cop6 and the effect of acidic or basic conditions on loop mutations in Cop4. Directed mutations of the H-α1 loop had a marked effect on the product profile of Cop3 and Cop4, while little to no change was shown in Cop6. Swapping of the Cop4 and Cop6 loops with one another was again shown to influence the product profile of Cop4, while the product profile of Cop6 remained identical to the wild-type enzyme. The loop mutations in Cop4 also implicate specific residues responsible for the pH sensitivity of the enzyme. These results affirm the role of the H-α1 loop in catalysis and provide a potential target to increase the product diversity of terpene synthases.

scholarly journals Evidence that cysteine-166 is the active-site nucleophile of Pseudomonas aeruginosa amidase: crystallization and preliminary X-ray diffraction analysis of the enzyme

1999 ◽  
Vol 340 (3) ◽  
pp. 711-714 ◽  
Author(s):  
Sebastien FARNAUD ◽  
Renée TATA ◽  
Maninder K. SOHI ◽  
Tommy WAN ◽  
Paul R. BROWN ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Diffraction Analysis ◽  
Active Site ◽  
Quaternary Structure ◽  
Cross Reactivity ◽  
Polyclonal Antiserum ◽  
Wild Type ◽  
X Ray Diffraction ◽  
Wild Type Enzyme ◽  
X Ray

Wild-type and site-specific mutants C166S and C166A (Cys-166 → Ser and Cys-166 → Ala respectively) of the amidase (acylamide amidohydrolase, EC 3.5.1.4) from Pseudomonas aeruginosa were expressed in Escherichia coli by using the vector pKK223-3. Both mutant proteins were catalytically inactive but showed complete cross-reactivity with polyclonal antiserum raised against the wild-type enzyme, as well as CD spectra identical with that of the wild-type enzyme, which were indicative of correct folding. Cys-166 is therefore implicated as the active-site nucleophile. Titration of free thiol groups with 5,5ʹ-dithiobis-(2-nitrobenzoic acid) indicated that Cys-166 is not a rapidly reacting residue. Crystals of both wild-type and C166S amidase grew with identical, rhombohedral morphology; X-ray diffraction analysis established the unit cell dimensions (a = b = c = 84 Å; α = β = γ = 75 °) and space group (R3 or R32). These results imply a quaternary structure of six subunits, with most probably 32 symmetry; the existence of a hexameric structure was supported by molecular mass determinations based on gel filtration and electrophoretic mobility.

scholarly journals Catalytic Dyad Residues His41 and Cys145 Impact the Catalytic Activity and Overall Conformational Fold of the Main SARS-CoV-2 Protease 3-Chymotrypsin-Like Protease

2021 ◽  
Vol 9 ◽  
Author(s):  
Juliana C. Ferreira ◽  
Samar Fadl ◽  
Adrian J. Villanueva ◽  
Wael M. Rabeh
Keyword(s):  
Catalytic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Oligomeric State ◽  
Wild Type Enzyme ◽  
Complete Inactivation ◽  
Catalytic Function ◽  
Catalytic Inactivation ◽  
Catalytic Dyad

Coronaviruses are responsible for multiple pandemics and millions of deaths globally, including the current pandemic of coronavirus disease 2019 (COVID-19). Development of antivirals against coronaviruses, including the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) responsible for COVID-19, is essential for containing the current and future coronavirus outbreaks. SARS-CoV-2 proteases represent important targets for the development of antivirals because of their role in the processing of viral polyproteins. 3-Chymotrypsin-like protease (3CLpro) is one such protease. The cleavage of SARS-CoV-2 polyproteins by 3CLpro is facilitated by a Cys145–His41 catalytic dyad. We here characterized the catalytic roles of the cysteine–histidine pair for improved understanding of the 3CLpro reaction mechanism, to inform the development of more effective antivirals against Sars-CoV-2. The catalytic dyad residues were substituted by site-directed mutagenesis. All substitutions tested (H41A, H41D, H41E, C145A, and C145S) resulted in a complete inactivation of 3CLpro, even when amino acids with a similar catalytic function to that of the original residues were used. The integrity of the structural fold of enzyme variants was investigated by circular dichroism spectroscopy to test if the catalytic inactivation of 3CLpro was caused by gross changes in the enzyme secondary structure. C145A, but not the other substitutions, shifted the oligomeric state of the enzyme from dimeric to a higher oligomeric state. Finally, the thermodynamic stability of 3CLpro H41A, H41D, and C145S variants was reduced relative the wild-type enzyme, with a similar stability of the H41E and C145A variants. Collectively, the above observations confirm the roles of His41 and Cys145 in the catalytic activity and the overall conformational fold of 3CLpro SARS-CoV-2. We conclude that the cysteine–histidine pair should be targeted for inhibition of 3CLpro and development of antiviral against COVID-19 and coronaviruses.

scholarly journals Combined effects of double mutations on catalytic activity and structural stability contribute to clinical manifestations of glucose-6-phosphate dehydrogenase deficiency

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Phonchanan Pakparnich ◽  
Sirapapha Sudsumrit ◽  
Mallika Imwong ◽  
Teeraporn Suteewong ◽  
Kamonwan Chamchoy ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Structural Stability ◽  
Clinical Manifestations ◽  
Enzyme Deficiency ◽  
Combined Effects ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Catalytically Active ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Structurally Stable

AbstractGlucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans, affecting ~ 500 million worldwide. A detailed study of the structural stability and catalytic activity of G6PD variants is required to understand how different mutations cause varying degrees of enzyme deficiency, reflecting the response of G6PD variants to oxidative stress. Furthermore, for G6PD double variants, investigating how two mutations jointly cause severe enzyme deficiency is important. Here, we characterized the functional and structural properties of nine G6PD variants: G6PD Gaohe, G6PD Mahidol, G6PD Shoklo, G6PD Canton, G6PD Kaiping, G6PD Gaohe + Kaiping, G6PD Mahidol + Canton, G6PD Mahidol + Kaiping and G6PD Canton + Kaiping. All variants were less catalytically active and structurally stable than the wild type enzyme, with G6PD double mutations having a greater impact than single mutations. G6PD Shoklo and G6PD Canton + Kaiping were the least catalytically active single and double variants, respectively. The combined effects of two mutations were observed, with the Canton mutation reducing structural stability and the Kaiping mutation increasing it in the double mutations. Severe enzyme deficiency in the double mutants was mainly determined by the trade-off between protein stability and catalytic activity. Additionally, it was demonstrated that AG1, a G6PD activator, only marginally increased G6PD enzymatic activity and stability.

Contribution of the C30/C75 disulfide bond to the biological properties of onconase

2008 ◽  
Vol 389 (8) ◽  
Author(s):  
Gerard Torrent ◽  
Antoni Benito ◽  
Jessica Castro ◽  
Marc Ribó ◽  
Maria Vilanova
Keyword(s):  
Catalytic Activity ◽  
Disulfide Bond ◽  
Chemotherapeutic Agent ◽  
Conformational Stability ◽  
Biological Properties ◽  
Redox Conditions ◽  
Wild Type ◽  
Ribonuclease Inhibitor ◽  
Different Types

Abstract Onconase, a member of the pancreatic type ribonuclease family, is currently used as a chemotherapeutic agent for the treatment of different types of cancer. It is widely accepted that one of the properties that renders this enzyme cytotoxic is its ability to evade the cytosolic ribonuclease inhibitor (RI). In the present work, we produced and characterized an onconase variant that lacks the disulfide bond C30/C75. This variant mimics the stable unfolding intermediate des(30–75) produced in the reductive unfolding pathway of onconase. We found that the reduction of the C30/C75 disulfide bond does not significantly alter the cytotoxic properties of onconase, although the variant possesses a notably reduced conformational stability. Interestingly, both its catalytic activity and its ability to evade RI are comparable to wild-type onconase under mild reductive conditions in which the three disulfide containing intermediate des(30–75) is present. These results suggest that the C30/C75 disulfide bond could easily be reduced under physiological redox conditions.

scholarly journals Construction and characterization of secreted and chimeric transmembrane forms of Drosophila acetylcholinesterase: a large truncation of the C-terminal signal peptide does not eliminate glycoinositol phospholipid anchoring.

1996 ◽  
Vol 7 (4) ◽  
pp. 595-611 ◽  
Author(s):  
J P Incardona ◽  
T L Rosenberry
Keyword(s):  
Catalytic Activity ◽  
Cell Surface ◽  
Signal Peptide ◽  
Cell Biology ◽  
Quaternary Structure ◽  
Cultured Cells ◽  
Biochemical Properties ◽  
Wild Type ◽  
In Vivo Experiments

Despite advances in understanding the cell biology of glycoinositol phospholipid (GPI)-anchored proteins in cultured cells, the in vivo functions of GPI anchors have remained elusive. We have focused on Drosophila acetylcholinesterase (AChE) as a model GPI-anchored protein that can be manipulated in vivo with sophisticated genetic techniques. In Drosophila, AChE is found only as a GPI-anchored G2 form encoded by the Ace locus on the third chromosome. To pursue our goal of replacing wild-type GPI-anchored AChE with forms that have alternative anchor structures in transgenic files, we report the construction of two secreted forms of Drosophila AChE (SEC1 and SEC2) and a chimeric form (TM-AChE) anchored by the transmembrane and cytoplasmic domains of herpes simplex virus type 1 glycoprotein C. To confirm that the biochemical properties of these AChEs were unchanged from GPI-AChE except as predicted, we made stably transfected Drosophila Schneider Line 2(S2) cells expressing each of the four forms. TM-AChE, SEC1, and SEC2 had the same catalytic activity and quaternary structure as wild type. TM-AChE was expressed as an amphiphilic membrane-bound protein resistant to an enzyme that cleaves GPI-AChE (phosphatidylinositol-specific phospholipase C), and the same percentage of TM-AChE and GPI-AChE was on the cell surface according to immunofluorescence and pharmacological data. SEC1 and SEC2 were constructed by truncating the C-terminal signal peptide initially present in GPI-AChE: in SEC1 the last 25 residues of this 34-residue peptide were deleted while in SEC2 the last 29 were deleted. Both SEC1 and SEC2 were efficiently secreted and are very stable in culture medium; with one cloned SEC1-expressing line, AChE accumulated to as high as 100 mg/liter. Surprisingly, 5-10% of SEC1 was attached to a GPI anchor, but SEC2 showed no GPI anchoring. Since no differences in catalytic activity were observed among the four AChEs, and since the same percentage of GPI-AChE and TM-AChE were on the cell surface, we contend that in vivo experiments in which GPI-AChE is replaced can be interpreted solely on the basis of the altered anchoring domain.

scholarly journals Site-directed mutagenesis of cysteine-195 in isocitrate lyase from Escherichia coli ML308

1995 ◽  
Vol 305 (1) ◽  
pp. 239-244 ◽  
Author(s):  
A G S Robertson ◽  
H G Nimmo
Keyword(s):  
Escherichia Coli ◽  
Isocitrate Lyase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Active Site Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Native Page ◽  
Mutant Proteins

Cysteine-195 was previously identified as a probable active site residue in isocitrate lyase (ICL) from Escherichia coli ML308 [Nimmo, Douglas, Kleanthous, Campbell and MacKintosh (1989) Biochem. J. 261, 431-435]. This residue was replaced with serine and alanine residues by site-directed mutagenesis. The mutated genes expressed proteins with low but finite ICL activity, which co-migrated with wild-type ICL on both SDS/ and native PAGE. The mutant proteins were purified and characterized. Fluorimetry and c.d. in both the near- and the far-u.v. regions showed no differences between the mutants and wild-type ICL, indicating that the conformations of the three enzymes were very similar. ICL C195A (Cys-195-->Ala) and C195S (Cys-195-->Ser) showed 8.4-fold and 3.6-fold increases in the Km for isocitrate, while their kcat. values showed 30- and 100-fold decreases respectively. The effect of pH on the kinetic properties of the wild-type and mutant ICLs was investigated. The results showed that the response of the mutant enzymes to pH was simpler than that of the wild-type. For the mutants, ionisation of a group with a pKa of approx. 7.8 affected the Km for isocitrate and kcat.. For the wild-type enzyme, these parameters were affected by the ionization of two or more groups, one of which is presumed to by cysteine-195. The results are consistent with the view that the previously identified group with a pKa of 7.1 whose ionization affects the reaction of ICL by iodoacetate is cysteine-195 itself.

scholarly journals Creation of a fully active, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase by the deletion of a membrane-spanning domain

1999 ◽  
Vol 23 (2) ◽  
pp. 231-239 ◽  
Author(s):  
JL Thomas ◽  
BW Evans ◽  
G Blanco ◽  
JI Mason ◽  
RC Strickler
Keyword(s):  
Deletion Mutant ◽  
Quaternary Structure ◽  
Hydroxysteroid Dehydrogenase ◽  
Type I ◽  
Wild Type ◽  
Membrane Domain ◽  
Wild Type Enzyme ◽  
Membrane Bound ◽  
Cell Cytosol ◽  
Specific Activities

Human 3beta-hydroxysteroid dehydrogenase/steroid Delta(5)-Delta(4)-isomerase (3beta-HSD/isomerase) is a bifunctional, single enzyme protein that is membrane-bound in the endoplasmic reticulum (microsomes) and mitochondria of cells in the placenta (type I) and in the adrenals and gonads (type II). Two membrane-binding domains (residues 72-89 and 283-310) have been predicted by analyses of hydrophobicity in the type I and II isoenzymes (90% regional homology). These putative membrane domains were deleted in the cDNA by PCR-based mutagenesis, and the two mutant enzymes were expressed by baculovirus in insect Sf9 cells. Differential centrifugation of the Sf9 cell homogenate containing the 283-310 deletion mutant revealed that 94% of the 3beta-HSD and isomerase activities were in the cell cytosol, 6% of the activities were in the microsomes, and no activity was in the mitochondria. This is the opposite of the subcellular distribution of the wild-type enzyme with 94% of the activities in the microsomes and mitochondria and only 6% activity in the cytosol. The organelle distribution of the 72-89 deletion mutant lies between these two extremes with 72% of the enzyme activity in the cytosol and 28% in the microsomes/mitochondria. The integrity of the subcellular organelle preparations was confirmed by electron microscopy. Western immunoblots confirmed the presence of the 283-310 deletion mutant enzyme and the absence of the wild-type enzyme in the insect cell cytosol. The unpurified, cytosolic 383-310 deletion mutant exhibited 3beta-HSD (22 nmol/min per mg) and isomerase (33 nmol/min per mg) specific activities that were comparable with those of the membrane-bound, wild-type enzyme. The isomerase reaction of the cytosolic 283-311 deletion mutant requires activation by NADH just like the isomerase of the microsomal or mitochondrial wild-type enzyme. In contrast, the 72-89 deletion mutant had low 3beta-HSD and isomerase specific activities that were only 12% of the wild-type levels. This innovative study identifies the 283-310 region as the critical membrane domain of 3beta-HSD/isomerase that can be deleted without compromising enzyme function. The shorter 72-89 region is also a membrane domain, but deletion of this NH(2)-terminal region markedly diminishes the enzyme activities. Purification of the active, cytosolic 283-310 deletion mutant will produce a valuable tool for crystallographic studies that may ultimately determine the tertiary/quaternary structure of this key steroidogenic enzyme.

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