scholarly journals Paenibacillus sp. TS12 glucosylceramidase: kinetic studies of a novel sub-family of family 3 glycosidases and identification of the catalytic residues

2004 ◽  
Vol 378 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Krisztina PAAL ◽  
Makoto ITO ◽  
Stephen G. WITHERS
Keyword(s):  
Kinetic Studies ◽  
Acid Base ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Dependent Inactivation ◽  
Paenibacillus Sp ◽  
Continuous Assay ◽  
Reduced Activity ◽  
Enzyme Intermediate

GCase (glucosylceramidase) from Paenibacillus sp. TS12, a family 3 glycosidase, hydrolyses the β-glycosidic linkage of glucosylceramide with retention of anomeric configuration via a two-step, double-displacement mechanism. Two carboxyl residues are essential for catalysis, one functioning as a nucleophile and the other as a general acid/base catalyst. p-Nitrophenyl β-d-glucopyranoside [Km=0.27±0.02 mM and kcat/Km=(2.1±0.2)×106 M−1·s−1] and 2,4-dinitrophenyl β-d-glucopyranoside [Km=0.16±0.02 mM and kcat/Km=(2.9±0.4)×106 M−1·s−1] were used for continuous assay of the enzyme. The dependence of kcat (and kcat/Km) on pH revealed a dependence on a group of pKa≤7.8 in the enzyme–substrate complex which must be protonated for catalysis. Incubation of GCase with 2,4-dinitrophenyl 2-deoxy-2-fluoro-β-d-glucopyranoside caused time-dependent inactivation (Ki=2.4±0.7 mM and ki=0.59±0.05 min−1) due to the accumulation of a trapped glycosyl–enzyme intermediate. Electrospray ionization MS analysis of the peptic digest of this complex showed that the enzyme was covalently labelled by the reagent at Asp-223, consistent with its role as nucleophile. A mutant modified at this residue (D223G) showed substantially reduced activity compared with the wild type (>104), but this activity could be partially restored by addition of formate as an external nucleophile. Kinetic analysis of the mutant E411A indicated that Glu-411 serves as the general acid/base catalytic residue since this mutant was pH-independent and since considerable GCase activity was restored upon addition of azide to E411A, along with formation of a glycosyl azide product.

scholarly journals The effect of pH on the kinetics of arylsulphatases A and B

1983 ◽  
Vol 213 (3) ◽  
pp. 603-607 ◽  
Author(s):  
C O'Fagain ◽  
B M Butler ◽  
T J Mantle
Keyword(s):  
Enzyme Activity ◽  
Rat Liver ◽  
Substrate Binding ◽  
Acid Base ◽  
Effect Of Ph ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
General Acid ◽  
Kinetics Of

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.

Kinetic studies of prothrombin activation: effect of factor Va and phospholipids on formation of the enzyme-substrate complex

Biochemistry ◽  
1984 ◽  
Vol 23 (20) ◽  
pp. 4557-4564 ◽  
Author(s):  
Jan L. M. L. Van Rijn ◽  
Jose W. P. Govers-Riemslag ◽  
Robert F. A. Zwaal ◽  
Jan Rosing
Keyword(s):  
Kinetic Studies ◽  
Activation Effect ◽  
Substrate Complex ◽  
Factor Va ◽  
Enzyme Substrate ◽  
Prothrombin Activation

scholarly journals Kinetic specificity in papain-catalysed hydrolyses

1971 ◽  
Vol 124 (1) ◽  
pp. 107-115 ◽  
Author(s):  
G. Lowe ◽  
Y. Yuthavong
Keyword(s):  
Protein Conformation ◽  
Proteolytic Enzyme ◽  
Peptide Bond ◽  
Acyl Residue ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Complex Models ◽  
Hydrophobic Amino Acids ◽  
Amplification Mechanism ◽  
Enzyme Intermediate

The specificity of the proteolytic enzyme, papain, for the peptide bond of the substrate adjacent to that about to be cleaved and for the acyl residue of some N-acylglycine derivatives is manifest almost exclusively in the formation of the acyl-enzyme from the enzyme–substrate complex. Models for the enzyme–substrate complex and acyl-enzyme intermediate are suggested that account for these observations. In particular it is suggested that the peptide bond of the substrate adjacent to that about to be cleaved, is bound in the cleft of the enzyme between the NH group of glycine-66 and the backbone C=O group of aspartic acid-158, and provides a sensitive amplification mechanism through which the specificity of the enzyme for hydrophobic amino acids such as l-phenylalanine is relayed. It is also suggested that the distortion in the enzyme–substrate complex and the binding of the peptide bond adjacent to that about to be cleaved are also linked and behave co-operatively, the distortion of the protein facilitating binding and the stronger binding facilitating distortion. The results imply that between the enzyme–substrate complex and the acyl-enzyme a relaxation of the protein conformation must occur.

scholarly journals Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

Biosensors ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 145
Author(s):  
Antonio Guerrieri ◽  
Rosanna Ciriello ◽  
Giuliana Bianco ◽  
Francesca De Gennaro ◽  
Silvio Frascaro
Keyword(s):  
Trichoderma Viride ◽  
Kinetic Studies ◽  
Pt Electrode ◽  
Allosteric Enzyme ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Influence ◽  
Ph Dependent ◽  
Kinetics Of ◽  
Allosteric Properties

The present study describes the kinetics of L-lysine-α-oxidase (LO) from Trichoderma viride immobilised by co-crosslinking onto the surface of a Pt electrode. The resulting amperometric biosensor was able to analyse L-lysine, thus permitting a simple but thorough study of the kinetics of the immobilised enzyme. The kinetic study evidenced that LO behaves in an allosteric fashion and that cooperativity is strongly pH-dependent. Not less important, experimental evidence shows that cooperativity is also dependent on substrate concentration at high pH and behaves as predicted by the Monod-Wyman-Changeux model for allosteric enzymes. According to this model, the existence of two different conformational states of the enzyme was postulated, which differ in Lys species landing on LO to form the enzyme–substrate complex. Considerations about the influence of the peculiar LO kinetics on biosensor operations and extracorporeal reactor devices will be discussed as well. Not less important, the present study also shows the effectiveness of using immobilised enzymes and amperometric biosensors not only for substrate analysis, but also as a convenient tool for enzyme kinetic studies.

scholarly journals KPC-2 β-lactamase Enables Carbapenem Antibiotic Resistance Through Fast Deacylation of the Covalent Intermediate

2020 ◽  
pp. jbc.RA120.015050
Author(s):  
Shrenik C Mehta ◽  
Ian M Furey ◽  
Orville A Pemberton ◽  
David M Boragine ◽  
Yu Chen ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
The Common ◽  
Covalent Intermediate ◽  
Hydrolysis Of ◽  
Carbapenem Antibiotic ◽  
Enzyme Intermediate

Serine active-site β-lactamases hydrolyze β-lactam antibiotics through formation of a covalent acyl-enzyme intermediate followed by deacylation via an activated water molecule. Carbapenem antibiotics are poorly hydrolyzed by most β-lactamases due to slow hydrolysis of the acyl-enzyme intermediate. However, the emergence of the KPC-2 carbapenemase has resulted in widespread resistance to these drugs, suggesting it operates more efficiently. Here, we investigated the unusual features of KPC-2 that enable this resistance. We show that KPC-2 has a 20,000-fold increased deacylation rate compared to the common TEM-1 β-lactamase. Further, kinetic analysis of active site alanine mutants indicates that carbapenem hydrolysis is a concerted effort involving multiple residues. Substitution of Asn170 greatly decreases the deacylation rate, but this residue is conserved in both KPC-2 and non-carbapenemase β-lactamases, suggesting it promotes carbapenem hydrolysis only in the context of KPC-2. X-ray structure determination of the N170A enzyme in complex with hydrolyzed imipenem suggests Asn170 may prevent the inactivation of the deacylating water by the 6α-hydroxyethyl substituent of carbapenems. In addition, the Thr235 residue, which interacts with the C3 carboxylate of carbapenems, also contributes strongly to the deacylation reaction. In contrast, mutation of the Arg220 and Thr237 residues decreases the acylation rate and, paradoxically, improves binding affinity for carbapenems. Thus, the role of these residues may be ground state destabilization of the enzyme-substrate complex or, alternatively, to ensure proper alignment of the substrate with key catalytic residues to facilitate acylation. These findings suggest modifications of the carbapenem scaffold to avoid hydrolysis by KPC-2 β-lactamase.

Synthesis and Kinase Phosphorylation of 4-Deoxy-D-threohexulose

1973 ◽  
Vol 51 (7) ◽  
pp. 969-972 ◽  
Author(s):  
Clifford Raymond Haylock ◽  
Keith Norman Slessor
Keyword(s):  
Lithium Aluminum Hydride ◽  
Aluminum Hydride ◽  
Kinetic Studies ◽  
Lithium Aluminum ◽  
Substrate Complex ◽  
Acetobacter Suboxydans ◽  
Enzyme Substrate ◽  
Kinase Phosphorylation ◽  
Hydrolysis Of ◽  
Yeast Hexokinase

Synthesis of the only unknown deoxyfructose, 4-deoxy-D-threohexulose, is reported. Its preparation involved reductive lithium aluminum hydride ring opening of 3,4-anhydro-1,2:5,6-di-O-isopropylidene- D-talitol, followed by hydrolysis of the resulting epimeric deoxy diisopropylidene hexitols and selective Acetobacter suboxydans oxidation of 3-deoxy-D-arabinohexitol. Kinetic studies using 4-deoxy-D-threohexulose as substrate for yeast hexokinase support the premise that the C-4 hydroxyl is a binding group in formation of the enzyme–substrate complex. Enzymatic synthesis of 4-deoxy-D-threohexulose 6-phosphate and 4-deoxy-D-threohexulose 1,6-diphosphate has been achieved in low yield from 4-deoxy-D-threohexulose.

Kinetic Studies of the Flagellar Movement of Sea-Urchin Spermatozoa

1969 ◽  
Vol 50 (1) ◽  
pp. 203-222
Author(s):  
M. E. J. HOLWILL
Keyword(s):  
Dielectric Constant ◽  
Ionic Strength ◽  
Sea Urchin ◽  
Activation Parameters ◽  
Kinetic Studies ◽  
Basic Part ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Glycerinated Model ◽  
Rate Limiting

1. The movements of intact and glycerinated spermatozoa from two species of sea-urchin were examined in the range of temperature between 5 and 30° C. The variations of the frequency of the glycerinated spermatozoa with changing pH, dielectric constant and ionic strength were examined. 2. From the temperature studies values for activation entropies and enthalpies were obtained that pertain to the chemical reaction which limits the frequency. 3. Within the limits of experimental error the activation parameters are identical for the intact spermatozoa and for the glycerinated model of the same species. The results are consistent with the hypothesis that the rate-limiting reaction is the breakdown of an enzyme-substrate complex. 4. The pH studies suggest that neither the acidic nor the basic part of the enzyme is involved in complex formation but that both participate in the breakdown of the complex. 5. The results obtained from the studies of dielectric constant are interpreted in terms of a model for the breakdown of the enzyme-substrate complex involving the separation of several spherical charges. 6. The studies of pH and ionic strength suggest that both 14 S and 30 S dynein participate in the mechano-chemical process responsible for bending the flagellum.

scholarly journals Trapping the acyl-enzyme intermediate in β-lactamase I catalysis

1989 ◽  
Vol 263 (3) ◽  
pp. 905-912 ◽  
Author(s):  
S J Cartwright ◽  
A K Tan ◽  
A L Fink
Keyword(s):  
Enzyme Digestion ◽  
Covalent Attachment ◽  
Beta Lactamase ◽  
Peptide Fragment ◽  
Aqueous Methanol ◽  
Fragment Analysis ◽  
Substrate Complex ◽  
Rate Determining Step ◽  
Enzyme Substrate ◽  
Enzyme Intermediate

Cryoenzymology techniques were used to facilitate trapping an acyl-enzyme intermediate in beta-lactamase I catalysis. The enzyme (from Bacillus cereus) was investigated in aqueous methanol cryosolvents over the 25 to -75 degrees C range, and was stable and functional in 70% (v/v) methanol at and below 0 degree C. The value of kcat. decreased linearly with increasing methanol concentration, suggesting that water is a reactant in the rate-determining step. In view of this, the lack of incorporation of methanol into the product means that the water molecule involved in the deacylation is shielded from bulk solvent in the enzyme-substrate complex. From the lack of adverse effects of methanol on the catalytic and structural properties of the enzyme we conclude that 70% methanol is a satisfactory cryosolvent system for beta-lactamase I. The acyl-enzyme intermediate from the reaction with 6-beta-(furylacryloyl)amidopenicillanic acid was accumulated in steady-state experiments at -40 degrees C and the reaction was quenched by lowering the pH to 2. H.p.l.c. experiments showed covalent attachment of the penicillin to the enzyme. Digestion by pepsin and trypsin yielded a single labelled peptide fragment; analysis of this peptide was consistent with Ser-70 as the site of attachment.

The roles of Tyr91 and Lys162 in general acid–base catalysis in the pigeon NADP+-dependent malic enzyme

2008 ◽  
Vol 411 (3) ◽  
pp. 467-473 ◽  
Author(s):  
Cheng-Chin Kuo ◽  
Kuan-Yu Lin ◽  
Yau-Jung Hsu ◽  
Shu-Yu Lin ◽  
Yu-Tsen Lin ◽  
...  
Keyword(s):  
Malic Enzyme ◽  
Kinetic Studies ◽  
Base Catalysis ◽  
Site Directed Mutagenesis ◽  
Decarboxylase Activity ◽  
Wild Type ◽  
Acid Base ◽  
Enzymatic Mechanism ◽  
General Acid ◽  
Steady State Kinetic

The role of general acid–base catalysis in the enzymatic mechanism of NADP+-dependent malic enzyme was examined by detailed steady-state kinetic studies through site-directed mutagenesis of the Tyr91 and Lys162 residues in the putative catalytic site of the enzyme. Y91F and K162A mutants showed approx. 200- and 27000-fold decreases in kcat values respectively, which could be partially recovered with ammonium chloride. Neither mutant had an effect on the partial dehydrogenase activity of the enzyme. However, both Y91F and K162A mutants caused decreases in the kcat values of the partial decarboxylase activity of the enzyme by approx. 14- and 3250-fold respectively. The pH-log(kcat) profile of K162A was found to be different from the bell-shaped profile pattern of wild-type enzyme as it lacked a basic pKa value. Oxaloacetate, in the presence of NADPH, can be converted by malic enzyme into L-malate by reduction and into enolpyruvate by decarboxylation activities. Compared with wild-type, the K162A mutant preferred oxaloacetate reduction to decarboxylation. These results are consistent with the function of Lys162 as a general acid that protonates the C-3 of enolpyruvate to form pyruvate. The Tyr91 residue could form a hydrogen bond with Lys162 to act as a catalytic dyad that contributes a proton to complete the enol–keto tautomerization.

scholarly journals Kinetic Studies on the Enzyme-substrate Complex Formation of p-Hydroxybenzoate Hydroxylase by the Stopped-flow Method

1972 ◽  
Vol 27 (10) ◽  
pp. 1172-1175 ◽  
Author(s):  
Naoki Higashi ◽  
Hirohumi Shoun ◽  
Keiji Yano ◽  
Κει Arima ◽  
Keitaro Hiromi
Keyword(s):  
Complex Formation ◽  
Reduction Rate ◽  
Kinetic Studies ◽  
Stopped Flow ◽  
Apparent Velocity ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Hydroxybenzoate Hydroxylase ◽  
Anaerobic Reduction

The spectrophotometric and spectrofluorometric investigations of the enzyme-substrate complex formation of p-hydroxybenzoate hydroxylase was made by the stopped-flow technique. The apparent velocity of the formation of the enzyme-substrate complex (the velocity of the absorbance change in visible and UV regions, and the velocity of the quenching of the fluorescence intensity in the FAD moiety of the holoenzyme by the substrate) was rapid enough to explain the maximal overall velocity (72 sec-1) or the activated anaerobic reduction rate (kredmax= 200 sec-1). The results were consistent with a two-step mechanism involving a rapid bimolecular association of enzyme and substrate, and a slower follow-up unimolecular process.

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