scholarly journals Heparin enhances the catalytic activity of des-ETW-thrombin

1996 ◽  
Vol 315 (1) ◽  
pp. 77-83 ◽  
Author(s):  
Christopher A. GOODWIN ◽  
John J. DEADMAN ◽  
Bernard F. LE BONNIEC ◽  
Said ELGENDY ◽  
Vijay V. KAKKAR ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Positive Influence ◽  
Heparin Binding ◽  
Wild Type ◽  
Pentosan Polysulphate ◽  
Initial Interaction ◽  
Sulphated Glycosaminoglycans ◽  
Hydrolysis Of ◽  
Increased Activity ◽  
Primary Specificity

The thrombin mutant, des-ETW-thrombin, lacking Glu146, Thr147 and Trp148 within a unique insertion loop located at the extreme end of the primary specificity pocket, has been shown previously to exhibit reduced catalytic activity with respect to macromolecular and synthetic thrombin substrates and reduced or enhanced susceptibility to inhibition. Investigation of the hydrolysis of peptidyl p-nitroanilide substrates by des-ETW-thrombin showed increased activity in the presence of heparin and other sulphated glycosaminoglycans. No effect was observed upon the activity of wild-type thrombin. Heparin was found to decrease the Km for cleavage of four thrombin-specific substrates by des-ETW-thrombin, by 3–4-fold. Similarly, pentosan polysulphate (PPS) decreased the Km with these substrates by 8–10-fold. Heparin also increased the rate of inhibition of des-ETW-thrombin by antithrombin III and D-phenylalanyl-prolyl-arginylchloromethane (PPACK). The inhibition of des-ETW-thrombin by a number of thrombin-specific peptide boronic acids also showed significant reduction in the final Ki in the presence of heparin, due to reduction in the off-rate. A peptide analogue of a sequence of hirudin which binds thrombin tightly to exosite 1 (fibrinogen recognition site) potentiated the activity of des-ETW-thrombin against peptide p-nitroanilide substrates in a manner similar to heparin. The Ki for the inhibition of des-ETW-thrombin by p-aminobenzamidine was decreased by these ligands from 9.7 mM to 7.5 mM, 5.1 mM and 2.5 mM in the presence of heparin, hirudin peptide and PPS respectively, suggesting the increased catalytic activity is due to enhanced access to the primary specificity pocket. The positive influence of these ligands on des-ETW-thrombin was reversed in the presence of ATP or ADP; the latter has previously been shown to inhibit thrombin activity by blocking initial interaction with fibrinogen at exosite 1. Because the effect of heparin and PPS is similar to that of hirudin peptide, it is proposed that the most likely mechanism is that binding at the heparin-binding site (thrombin exosite 2) facilitates interaction at exosite 1 causing a conformational change which partially corrects the defective ground-state binding of the mutant thrombin. Although no effect was observed upon the activity of wild-type thrombin, our findings do provide further evidence of an allosteric property of thrombin which may control the geometry of, and access to, the primary specificity pocket.

scholarly journals Mutating His29, His125, His133 or His158 abolishes glycosylphosphatidylinositol-specific phospholipase D catalytic activity

2005 ◽  
Vol 391 (2) ◽  
pp. 285-289 ◽  
Author(s):  
Nandita S. Raikwar ◽  
Rosario F. Bowen ◽  
Mark A. Deeg
Keyword(s):  
Catalytic Activity ◽  
Phospholipase D ◽  
Catalytic Domain ◽  
Computer Modelling ◽  
Critical Role ◽  
Biochemical Properties ◽  
Catalytic Site ◽  
Wild Type ◽  
Histidine Residues ◽  
Hydrolysis Of

Glycosylphosphatidylinositol (GPI)-specific phospholipase D (GPI-PLD) specifically cleaves GPIs. This phospholipase D is a secreted protein consisting of two domains: an N-terminal catalytic domain and a predicted C-terminal β-propeller. Although the biochemical properties of GPI-PLD have been extensively studied, its catalytic site has not been identified. We hypothesized that a histidine residue(s) may play a critical role in the catalytic activity of GPI-PLD, based on the observations that (i) Zn2+, which utilizes histidine residues for binding, is required for GPI-PLD catalytic activity, (ii) a phosphohistidine intermediate is involved in phospholipase D hydrolysis of phosphatidylcholine, (iii) computer modelling suggests a catalytic site containing histidine residues, and (iv) our observation that diethyl pyrocarbonate, which modifies histidine residues, inhibits GPI-PLD catalytic activity. Individual mutation of the ten histidine residues to asparagine in the catalytic domain of murine GPI-PLD resulted in three general phenotypes: not secreted or retained (His56 or His88), secreted with catalytic activity (His34, His81, His98 or His219) and secreted without catalytic activity (His29, His125, His133 or His158). Changing His133 but not His29, His125 or His158 to Cys resulted in a mutant that retained catalytic activity, suggesting that at least His133 is involved in Zn2+ binding. His133 and His158 also retained the biochemical properties of wild-type GPI-PLD including trypsin cleavage pattern and phosphorylation by protein kinase A. Hence, His29, His125, His133 and His158 are required for GPI-PLD catalytic activity.

Immobilized Nanoparticles-Mediated Enzymatic Hydrolysis of Cellulose for Clean Sugar Production: A Novel Approach

2019 ◽  
Vol 15 (3) ◽  
pp. 296-303 ◽  
Author(s):  
Swapnil Gaikwad ◽  
Avinash P. Ingle ◽  
Silvio Silverio da Silva ◽  
Mahendra Rai
Keyword(s):  
Catalytic Activity ◽  
Enzymatic Hydrolysis ◽  
Immobilized Enzyme ◽  
Free Enzyme ◽  
Magnetic Nanomaterials ◽  
Sugar Production ◽  
Cellulase Enzyme ◽  
Enzymatic Hydrolysis Of Cellulose ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

Background: Enzymatic hydrolysis of cellulose is an expensive approach due to the high cost of an enzyme involved in the process. The goal of the current study was to apply magnetic nanomaterials as a support for immobilization of enzyme, which helps in the repeated use of immobilized enzyme for hydrolysis to make the process cost-effective. In addition, it will also provide stability to enzyme and increase its catalytic activity. Objective: The main aim of the present study is to immobilize cellulase enzyme on Magnetic Nanoparticles (MNPs) in order to enable the enzyme to be re-used for clean sugar production from cellulose. Methods: MNPs were synthesized using chemical precipitation methods and characterized by different techniques. Further, cellulase enzyme was immobilized on MNPs and efficacy of free and immobilized cellulase for hydrolysis of cellulose was evaluated. Results: Enzymatic hydrolysis of cellulose by immobilized enzyme showed enhanced catalytic activity after 48 hours compared to free enzyme. In first cycle of hydrolysis, immobilized enzyme hydrolyzed the cellulose and produced 19.5 ± 0.15 gm/L of glucose after 48 hours. On the contrary, free enzyme produced only 13.7 ± 0.25 gm/L of glucose in 48 hours. Immobilized enzyme maintained its stability and produced 6.15 ± 0.15 and 3.03 ± 0.25 gm/L of glucose in second and third cycle, respectively after 48 hours. Conclusion: This study will be very useful for sugar production because of enzyme binding efficiency and admirable reusability of immobilized enzyme, which leads to the significant increase in production of sugar from cellulosic materials.

Yeast cells lacking 5'-->3' exoribonuclease 1 contain mRNA species that are poly(A) deficient and partially lack the 5' cap structure

1993 ◽  
Vol 13 (8) ◽  
pp. 4826-4835
Author(s):  
C L Hsu ◽  
A Stevens
Keyword(s):  
Full Length ◽  
Yeast Cells ◽  
Mrna Turnover ◽  
Turnover Rates ◽  
Wild Type ◽  
Mrna Species ◽  
Turnover Process ◽  
Hydrolysis Of ◽  
Extension Analysis ◽  
Wild Type Yeast

Analysis of the slowed turnover rates of several specific mRNA species and the higher cellular levels of some of these mRNAs in Saccharomyces cerevisiae lacking 5'-->3' exoribonuclease 1 (xrn1 cells) has led to the finding that these yeast contain higher amounts of essentially full-length mRNAs that do not bind to oligo(dT)-cellulose. On the other hand, the length of mRNA poly(A) chains found after pulse-labeling of cells lacking the exoribonuclease, the cellular rate of synthesis of oligo(dT)-bound mRNA, and the initial rate of its deadenylation appeared quite similar to the same measurements in wild-type yeast cells. Examination of the 5' cap structure status of the poly(A)-deficient mRNAs by comparative analysis of the m7G content of poly(A)- and poly(A)+ RNA fractions of wild-type and xrn1 cells suggested that the xrn1 poly(A)- mRNA fraction is low in cap structure content. Further analysis of the 5' termini by measurements of the rate of 5'-->3' exoribonuclease 1 hydrolysis of specific full-length mRNA species showed that approximately 50% of the xrn1 poly(A)-deficient mRNA species lack the cap structure. Primer extension analysis of the 5' terminus of ribosomal protein 51A (RP51A) mRNA showed that about 30% of the poly(A)-deficient molecules of the xrn1 cells are slightly shorter at the 5' end. The finding of some accumulation of poly(A)-deficient mRNA species partially lacking the cap structure together with the reduction of the rate of mRNA turnover in cells lacking the enzyme suggest a possible role for 5'-->3' exoribonuclease 1 in the mRNA turnover process.

scholarly journals Influence of Water Activity on Protease Adsorbed on Biochar in Organic Solvents

2017 ◽  
Vol 6 (4) ◽  
pp. 96 ◽  
Author(s):  
Hidetaka Noritomi ◽  
Jumpei Nishigami ◽  
Nobuyuki Endo ◽  
Satoru Kato ◽  
Katsumi Uchiyama
Keyword(s):  
Thermal Stability ◽  
Catalytic Activity ◽  
Organic Solvents ◽  
Water Activity ◽  
Initial Rate ◽  
Butyl Ester ◽  
Nitrogen Atmosphere ◽  
Bamboo Charcoal ◽  
Influence Of Water ◽  
Hydrolysis Of

We have found that the organic solvent-resistance of Alpha-chymotrypsin (Alpha-CT) is enhanced by adsorbing Alpha-CT onto bamboo charcoal powder (BCP), which is obtained by pyrolyzing bamboo waste under nitrogen atmosphere, and is markedly dependent on the thermodynamic water activity (aw) in organic solvents. When BCP-adsorbed　Alpha-CT was immersed in acetonitrile at an appropriate water activity, it effectively enhanced the transesterification of N-acetyl-L-tyrosine ethyl ester (N-Ac-Tyr-OEt) with n-butanol (BuOH) to produce N-acetyl-L-tyrosine butyl ester (N-Ac-Tyr-OBu), compared to the hydrolysis of N-Ac-Tyr-OEt with water to give N-acetyl-L-tyrosine (N-Ac-Tyr-OH). When the water activity was 0.28, the initial rate of transesterification catalyzed by BCP-adsorbed Alpha-CT was about sixty times greater than that catalyzed by free Alpha-CT. Regarding the reaction selectivity which is defined as a ratio of the initial rate of transesterification to that of hydrolysis, BCP-adsorbed α-CT was much superior to free Alpha-CT. The catalytic activity of BCP-adsorbed Alpha-CT was markedly dependent on the reaction temperature. Furthermore, concerning the thermal stability at 50 oC, the half-life of BCP-adsorbed Alpha-CT exhibited 3.8-fold, compared to that of free Alpha-CT.

scholarly journals GUCY2D Gene Loss-of-Function Mutations Responsible for Leber Congenital Amaurosis 1

2019 ◽  
Author(s):  
Feng Xue ◽  
Tianying Wei ◽  
Junhui Sun ◽  
Yuqin Luo ◽  
Yanan Huo ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Guanylate Cyclase ◽  
Cyclic Guanosine Monophosphate ◽  
Guanosine Monophosphate ◽  
Loss Of Function ◽  
Wild Type ◽  
Leber Congenital Amaurosis ◽  
Cellular Location ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Abstract Background: Leber congenital amaurosis (LCA) is a group of severe congenital neurodegenerative diseases. Variants in guanylate cyclase 2D (GUCY2D), which encoded guanylate cyclase protein (ROS-GC1) associate with LCA1, accounting for 6–21% of all LCA cases. Methods: In this study, one family with LCA1 was recruited from China. A combination of next-generation sequencing (NGS) and Sanger sequencing was used for disease-causing mutations screening. Additionally, immunohistochemistry and HPLC-coupled tandem mass-spectrometry (HPLC-MS/MS) were used to confirm the cellular location and catalytic activity of ROS-GC1 mutants, respectively. Results: We found three novel mutations (c.139_139delC, c.835G>A and c.2783G>A) in GUCY2D gene. The results showed that mutation c.139_139delC results in a truncated protein and destroys the structure of ROS-GC1 protein. Mutations c.835G>A and c.2783G>A exert no effects on cellular location, whereas significantly reduce the catalytic activity of ROS-GC1. Conclusions: Our findings highlight the clinical range of LCA. Moreover we used HPLC-MS/MS to analyze the concentration of 3', 5'-cyclic guanosine monophosphate (cGMP), suggesting that HPLC-MS/MS can be an effective alternative method to evaluate the catalytic activity of wild type (wt) and mutant ROS-GC1.

scholarly journals Conditions of hydrolysis with a specific pair of endo- and exo-cellulases

2021 ◽  
Author(s):  
Yoshiki Kitano
Keyword(s):  
Synergistic Effect ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Amorphous Cellulose ◽  
Potential Synergy ◽  
Hydrolysis Of Cellulose ◽  
Potential Alternative ◽  
Specific Pair ◽  
Hydrolysis Of ◽  
Increased Activity

Enzymatic hydrolysis of cellulose is a technology involved in the production of bioethanol, a potential alternative renewable energy. Many cellulases with endo- and exo- type of activity are known to hydrolyze cellulose synergistically. In this thesis, potential synergy between an endo-cellulase, Cel5B, with and without a carbohydrate- binding module (CBM6), and a new exo-cellulase, CBH1, from Trichoderma harzianum FP108 were examined during the hydrolysis of semi- crystalline cellulose (Avicel). Since CBM6 is recognized as having a high affinity for amorphous cellulose, it was hypothesized that this affinity could enhance the synergistic effect between the endo- and exo-cellulases by focusing the action to Cel5B+CBM6 on the amorphous regions of the Avicel substrate. The increased activity of Cel5B+CBM6 over Cel5B alone was confirmed. However, in contrast to our expectations, a synergistic effect was not observed between either endo- and exo-cellulase pairs. From the obtained hydrolysis yield, it was inferred that Cel5B+CBM6 may have exo-type activity that caused a competitive interaction with the exo-cellulase, which resulted in no synergy.

scholarly journals Human xylosyltransferase I and N-terminal truncated forms: functional characterization of the core enzyme

2006 ◽  
Vol 394 (1) ◽  
pp. 163-171 ◽  
Author(s):  
Sandra Müller ◽  
Jennifer Disse ◽  
Manuela Schöttler ◽  
Sylvia Schön ◽  
Christian Prante ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Binding Capacity ◽  
Functional Characterization ◽  
Terminal Region ◽  
Binding Motif ◽  
Heparin Binding ◽  
Loss Of Function ◽  
The Impact

Human XT-I (xylosyltransferase I; EC 2.4.2.26) initiates the biosynthesis of the glycosaminoglycan linkage region and is a diagnostic marker of an enhanced proteoglycan biosynthesis. In the present study, we have investigated mutant enzymes of human XT-I and assessed the impact of the N-terminal region on the enzymatic activity. Soluble mutant enzymes of human XT-I with deletions at the N-terminal domain were expressed in insect cells and analysed for catalytic activity. As many as 260 amino acids could be truncated at the N-terminal region of the enzyme without affecting its catalytic activity. However, truncation of 266, 272 and 273 amino acids resulted in a 70, 90 and >98% loss in catalytic activity. Interestingly, deletion of the single 12 amino acid motif G261KEAISALSRAK272 leads to a loss-of-function XT-I mutant. This is in agreement with our findings analysing the importance of the Cys residues where we have shown that C276A mutation resulted in a nearly inactive XT-I enzyme. Moreover, we investigated the location of the heparin-binding site of human XT-I using the truncated mutants. Heparin binding was observed to be slightly altered in mutants lacking 289 or 568 amino acids, but deletion of the potential heparin-binding motif P721KKVFKI727 did not lead to a loss of heparin binding capacity. The effect of heparin or UDP on the XT-I activity of all mutants was not significantly different from that of the wild-type. Our study demonstrates that over 80% of the nucleotide sequence of the XT-I-cDNA is necessary for expressing a recombinant enzyme with full catalytic activity.

Sign in / Sign up

Export Citation Format

Share Document