scholarly journals Fragment-derived modulators of an industrial β-glucosidase

2020 ◽  
Vol 477 (22) ◽  
pp. 4383-4395
Author(s):  
Eleni Makraki ◽  
John F. Darby ◽  
Marta G. Carneiro ◽  
James D. Firth ◽  
Alex Heyam ◽  
...  
Keyword(s):  
Binding Site ◽  
Glycoside Hydrolase ◽  
Mixed Model ◽  
Specific Binding ◽  
Fold Increase ◽  
Kinetic Assay ◽  
Nmr Spectrum ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Maximum Activation

A fragment screen of a library of 560 commercially available fragments using a kinetic assay identified a small molecule that increased the activity of the fungal glycoside hydrolase TrBgl2. An analogue by catalogue approach and detailed kinetic analysis identified improved compounds that behaved as nonessential activators with up to a 2-fold increase in maximum activation. The compounds did not activate the related bacterial glycoside hydrolase CcBglA demonstrating specificity. Interestingly, an analogue of the initial fragment inhibits both TrBgl2 and CcBglA, apparently through a mixed-model mechanism. Although it was not possible to determine crystal structures of activator binding to 55 kDa TrBgl2, solution NMR experiments demonstrated a specific binding site for the activator. A partial assignment of the NMR spectrum gave the identity of the amino acids at this site, allowing a model for TrBgl2 activation to be built. The activator binds at the entrance of the substrate-binding site, generating a productive conformation for the enzyme–substrate complex.

scholarly journals A novel β-xylosidase structure fromGeobacillus thermoglucosidasius: the first crystal structure of a glycoside hydrolase family GH52 enzyme reveals unpredicted similarity to other glycoside hydrolase folds

2014 ◽  
Vol 70 (5) ◽  
pp. 1366-1374 ◽  
Author(s):  
Giannina Espina ◽  
Kirstin Eley ◽  
Guillaume Pompidor ◽  
Thomas R. Schneider ◽  
Susan J. Crennell ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Glycoside Hydrolase ◽  
Thermophilic Bacterium ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Gene Encoding ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Family ◽  
Hydrolase Family

Geobacillus thermoglucosidasiusis a thermophilic bacterium that is able to ferment both C6 and C5 sugars to produce ethanol. During growth on hemicellulose biomass, an intracellular β-xylosidase catalyses the hydrolysis of xylo-oligosaccharides to the monosaccharide xylose, which can then enter the pathways of central metabolism. The gene encoding aG. thermoglucosidasiusβ-xylosidase belonging to CAZy glycoside hydrolase family GH52 has been cloned and expressed inEscherichia coli. The recombinant enzyme has been characterized and a high-resolution (1.7 Å) crystal structure has been determined, resulting in the first reported structure of a GH52 family member. A lower resolution (2.6 Å) structure of the enzyme–substrate complex shows the positioning of the xylobiose substrate to be consistent with the proposed retaining mechanism of the family; additionally, the deep cleft of the active-site pocket, plus the proximity of the neighbouring subunit, afford an explanation for the lack of catalytic activity towards the polymer xylan. Whilst the fold of theG. thermoglucosidasiusβ-xylosidase is completely different from xylosidases in other CAZy families, the enzyme surprisingly shares structural similarities with other glycoside hydrolases, despite having no more than 13% sequence identity.

scholarly journals The pyridoxal-binding site in pyridoxamine-pyruvate transaminase

1978 ◽  
Vol 169 (2) ◽  
pp. 429-432 ◽  
Author(s):  
J Hodsdon ◽  
H Kolb ◽  
E E Snell ◽  
R D Cole
Keyword(s):  
Binding Site ◽  
Pyridoxal Phosphate ◽  
Tryptic Digestion ◽  
Substrate Complex ◽  
Chromatography Analysis ◽  
Enzyme Substrate ◽  
Lysine Residues

The enzyme-substrate complex formed between pyridoxamine-pyruvate transaminase (EC 2.6.1.30) and pyridoxal was reduced with NaBH4. After carboxymethylation and tryptic digestion, pyridoxyl-lysine-containing peptides were isolated by a combination of Sephadex and Dowex 50 chromatography. Analysis of these peptides shows the structure around the pyridoxal-binding lysine residues to be Ala-Asp-Ile-Tyr-Val-Thr-Gly-Pro-Asx-Lys(Pxy)-Cys-Leu(Pro2, Gly2, Ala2, Met)(Thr, Leu2)Gly-Val-Ser-Glu-Arg. This structure differs from those found for the corresponding peptides from pyridoxal phosphate-dependent enzymes.

Protection of α-amylase from proteolysis by adsorption to feed components in vitro and in the porcine small intestine

2018 ◽  
Vol 58 (4) ◽  
pp. 640
Author(s):  
Anton M. Pluschke ◽  
Paulus G. M. Jochems ◽  
Barbara A. Williams ◽  
Michael J. Gidley
Keyword(s):  
Small Intestine ◽  
Digestive Enzymes ◽  
Specific Binding ◽  
Wheat Starch ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Food Components ◽  
Small Intestinal

The interactions between digestive enzymes and non-substrate feed components, and the impacts these have on enzyme activity, have rarely been studied. The aim of the present study was to determine the ability of granular wheat starch and whole porcine diets to protect porcine pancreatic α-amylase from proteolysis by trypsin both in vitro and in vivo. Granular wheat starch protected α-amylase from degradation in vitro by adsorbing trypsin and reducing its proteolytic activity. This protection was also found for a complete pig diet and corresponded to undetectable soluble-trypsin activity in the presence of the diet. Pancreatic α-amylase from small intestinal digesta of pigs was active from the duodenum to the ileum (~200–330 U/mL) irrespective of the addition of a protease inhibitor immediately after sampling, most likely due to binding with other food components protecting it from proteolysis. We conclude that non-specific binding between pancreatic digestive enzymes and food components may be competitive with enzyme–substrate complex formation, and therefore important in determining differences in the rate of digestion of macronutrients along the small intestine.

scholarly journals Activation mechanism and modification kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate

1998 ◽  
Vol 335 (1) ◽  
pp. 181-189 ◽  
Author(s):  
Jia-Wei WU ◽  
Zhi-Xin WANG
Keyword(s):  
Binding Energy ◽  
Dihydrofolate Reductase ◽  
Tertiary Structure ◽  
Chinese Hamster ◽  
Fold Increase ◽  
Activation Mechanism ◽  
Binary And Ternary Complexes ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Kinetics Of

Substrate effects on the activation kinetics of Chinese hamster dihydrofolate reductase by p-chloromercuribenzoate (pCMB) have been studied. On the basis of the kinetic equation of substrate reaction in the presence of pCMB, all modification kinetic constants for the free enzyme and enzyme–substrate binary and ternary complexes have been determined. The results of the present study indicate that the modification of Chinese hamster dihydrofolate reductase by pCMB shows single-phase kinetics, and that changes in the enzyme activity and tertiary structure proceed simultaneously during the modification process. Both substrates, NADPH and 7,8-dihydrofolate, protect dihydrofolate reductase against modification by pCMB. In the presence of a saturating concentration of NADPH, the value of kcat for 7,8-dihydrofolate in the enzyme-catalysed reaction increased four-fold on modification of Cys-6, accompanied by a two-fold increase in Km for the modified enzyme. The utilization of the binding energy of a group to increase kcat rather than reduce Km implies that the full binding energy of the group is not realized in the formation of the enzyme–substrate complex, but is used to stabilize the enzyme–transition-state complex.

The role of secondary alcoholic groups of D-galacturonan in its degradation with exo-D-galacturonanase

1980 ◽  
Vol 45 (2) ◽  
pp. 427-434 ◽  
Author(s):  
Kveta Heinrichová ◽  
Rudolf Kohn
Keyword(s):  
Acetyl Derivative ◽  
Competitive Inhibitor ◽  
Hydroxyl Groups ◽  
Pectic Acid ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Individual ◽  
Degradation Degree ◽  
Derivatives Of

The effect of exo-D-galacturonanase from carrot on O-acetyl derivatives of pectic acid of variousacetylation degree was studied. Substitution of hydroxyl groups at C(2) and C(3) of D-galactopyranuronic acid units influences the initial rate of degradation, degree of degradation and its maximum rate, the differences being found also in the time of limit degradations of the individual O-acetyl derivatives. Value of the apparent Michaelis constant increases with increase of substitution and value of Vmax changes. O-Acetyl derivatives act as a competitive inhibitor of degradation of D-galacturonan. The extent of the inhibition effect depends on the degree of substitution. The only product of enzymic reaction is D-galactopyranuronic acid, what indicates that no degradation of the terminal substituted unit of O-acetyl derivative of pectic acid takes place. Substitution of hydroxyl groups influences the affinity of the enzyme towards the modified substrate. The results let us presume that hydroxyl groups at C(2) and C(3) of galacturonic unit of pectic acid are essential for formation of the enzyme-substrate complex.

scholarly journals Spontaneous Cleavages of a Heterologous Protein, the CenA Endoglucanase of Cellulomonas fimi, in Escherichia coli

2021 ◽  
Vol 14 ◽  
pp. 117863612110246
Author(s):  
Cheuk Yin Lai ◽  
Ka Lun Ng ◽  
Hao Wang ◽  
Chui Chi Lam ◽  
Wan Keung Raymond Wong
Keyword(s):  
Escherichia Coli ◽  
Cellulolytic Bacterium ◽  
Systematic Screening ◽  
Cellulomonas Fimi ◽  
E Coli ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycosylation Sites ◽  
Endogenous Proteins ◽  
Cellulose Binding

CenA is an endoglucanase secreted by the Gram-positive cellulolytic bacterium, Cellulomonas fimi, to the environment as a glycosylated protein. The role of glycosylation in CenA is unclear. However, it seems not crucial for functional activity and secretion since the unglycosylated counterpart, recombinant CenA (rCenA), is both bioactive and secretable in Escherichia coli. Using a systematic screening approach, we have demonstrated that rCenA is subjected to spontaneous cleavages (SC) in both the cytoplasm and culture medium of E. coli, under the influence of different environmental factors. The cleavages were found to occur in both the cellulose-binding (CellBD) and catalytic domains, with a notably higher occurring rate detected in the former than the latter. In CellBD, the cleavages were shown to occur close to potential N-linked glycosylation sites, suggesting that these sites might serve as ‘attributive tags’ for differentiating rCenA from endogenous proteins and the points of initiation of SC. It is hypothesized that glycosylation plays a crucial role in protecting CenA from SC when interacting with cellulose in the environment. Subsequent to hydrolysis, SC would ensure the dissociation of CenA from the enzyme-substrate complex. Thus, our findings may help elucidate the mechanisms of protein turnover and enzymatic cellulolysis.

scholarly journals Conformational Analysis of the Enzyme-Substrate Complex in the Dehydrogenation of Sterols by Tetrahymena pyriformis

1971 ◽  
Vol 246 (3) ◽  
pp. 561-568 ◽  
Author(s):  
William R. Nes ◽  
P.A. Govinda Malya ◽  
Frank B. Mallory ◽  
Karen A. Ferguson ◽  
Josephine R. Landrey ◽  
...  
Keyword(s):  
Conformational Analysis ◽  
Tetrahymena Pyriformis ◽  
Substrate Complex ◽  
Enzyme Substrate

scholarly journals N 6-Methyladenosines in mRNAs reduce the accuracy of codon reading by transfer RNAs and peptide release factors

2021 ◽  
Vol 49 (5) ◽  
pp. 2684-2699
Author(s):  
Ka-Weng Ieong ◽  
Gabriele Indrisiunaite ◽  
Arjun Prabhakar ◽  
Joseph D Puglisi ◽  
Måns Ehrenberg
Keyword(s):  
Single Molecule ◽  
Stop Codon ◽  
Product Formation ◽  
Release Factors ◽  
Substrate Complex ◽  
Peptidyl Transfer ◽  
Enzyme Substrate ◽  
Peptide Release ◽  
Accuracy Ratio ◽  
Codon Reading

Abstract We used quench flow to study how N6-methylated adenosines (m6A) affect the accuracy ratio between kcat/Km (i.e. association rate constant (ka) times probability (Pp) of product formation after enzyme-substrate complex formation) for cognate and near-cognate substrate for mRNA reading by tRNAs and peptide release factors 1 and 2 (RFs) during translation with purified Escherichia coli components. We estimated kcat/Km for Glu-tRNAGlu, EF-Tu and GTP forming ternary complex (T3) reading cognate (GAA and Gm6AA) or near-cognate (GAU and Gm6AU) codons. ka decreased 10-fold by m6A introduction in cognate and near-cognate cases alike, while Pp for peptidyl transfer remained unaltered in cognate but increased 10-fold in near-cognate case leading to 10-fold amino acid substitution error increase. We estimated kcat/Km for ester bond hydrolysis of P-site bound peptidyl-tRNA by RF2 reading cognate (UAA and Um6AA) and near-cognate (UAG and Um6AG) stop codons to decrease 6-fold or 3-fold by m6A introduction, respectively. This 6-fold effect on UAA reading was also observed in a single-molecule termination assay. Thus, m6A reduces both sense and stop codon reading accuracy by decreasing cognate significantly more than near-cognate kcat/Km, in contrast to most error inducing agents and mutations, which increase near-cognate at unaltered cognate kcat/Km.

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