Studies on the mechanism of sheep liver cytosolic aldehyde dehydrogenase. The effect of pH on the aldehyde binding reactions and a re-examination of the problem of the site of proton release in the mechanism

Initial-rate measurements and stopped-flow spectrophotometric experiments over a wide range of pH implicate an enzyme group of pKa approximately 6.6 affecting the aldehyde binding reactions. It is possible, though not proved, that the group involved is the cysteine residue involved in catalysis. Stopped-flow fluorescence studies show that a group of pKa greater than 8.5 facilitates hydrolysis of the NADH-containing acyl-enzyme species. The identity of this group is quite unknown. Studies with 4-nitrobenzaldehyde show that this substrate gives marked substrate inhibition at quite low (less than 20 microM) concentrations. The mechanism of catalysis seems to be the same as for propionaldehyde oxidation. It is argued that proton release occurs with both substrates on hydrolysis of the NADH-containing acyl-enzyme and not before hydride transfer, as has been previously suggested [Bennett, Buckley & Blackwell (1982) Biochemistry 21, 4407-4413].

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Lead Molecules for Targeted Urease Inhibition: An Updated Review from 2010 -2018

Current Protein and Peptide Science ◽

10.2174/1389203720666190320170215 ◽

2019 ◽

Vol 20 (12) ◽

pp. 1158-1188 ◽

Cited By ~ 2

Author(s):

Ritu Kataria ◽

Anurag Khatkar

Keyword(s):

Enzyme Mechanism ◽

Bacillus Pasteurii ◽

Pathological Conditions ◽

Current Review ◽

Current Topic ◽

Wide Range ◽

Mechanism Of Catalysis ◽

Urease Inhibitory ◽

H Pylori ◽

Hydrolysis Of

The field of enzyme inhibition is a tremendous and quickly growing territory of research. Urease a nickel containing metalloenzyme found in bacteria, algae, fungi, and plants brings hydrolysis of urea and plays important role in environmental nitrogen cycle. Apart from this it was found to be responsible for many pathological conditions due to its presence in many microorganisms such as H. Pylori, a ureolytic bacteria having urease which elevates pH of gastric medium by hydrolyzing urea present in alimentary canal and help the bacteria to colonize and spread infection. Due to the infections caused by the various bacterial ureases such as Bacillus pasteurii, Brucella abortus, H. pylori, H. mustelae, Klebsiella aerogenes, Klebsiella tuberculosis, Mycobacterium tuberculosis, Pseudomonas putida, Sporosarcina pasteurii and Yersinia enterocolitica, it has been the current topic of today’s research. About a wide range of compounds from the exhaustive literature survey has been discussed in this review which is enveloped into two expansive classes, as Inhibitors from synthetic origin and Inhibitors from natural origin. Moreover active site details of enzyme, mechanism of catalysis of substrate by enzyme, uses of plant urease and its pathogenic behavior has been included in the current review. So, overall, this review article diagrams the current landscape of the developments in the improvements in the thriving field of urease inhibitory movement in medicinal chemistry from year 2010 to 2018, with an emphasis on mechanism of action of inhibitors that may be used for more development of recent and strong urease inhibitors and open up new doors for assist examinations in a standout amongst the most lively and promising regions of research.

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THE TRYPSIN CATALYZED HYDROLYSIS OF p-NITROPHENYL ACETATE

Canadian Journal of Chemistry ◽

10.1139/v59-102 ◽

1959 ◽

Vol 37 (4) ◽

pp. 751-759 ◽

Cited By ~ 27

Author(s):

James A. Stewart ◽

Ludovic Ouellet

Keyword(s):

Active Site ◽

Initial Rate ◽

Competitive Inhibition ◽

Early Stage ◽

Stopped Flow ◽

Hydrogen Ions ◽

Experimental Conditions ◽

Influence Of Ph ◽

Kinetics Of ◽

Hydrolysis Of

The hydrolysis of p-nitrophenyl acetate (NPA) by trypsin has been investigated in the early stage of the reaction using stopped-flow techniques. The influence of pH on the initial rate suggests competitive inhibition of the active site of the enzyme by hydrogen ions. The dissociation constant of the enzyme obtained from the kinetics of this reaction (pK = 6.9) indicates possible catalysis by an ammo group or an imidazole group of the enzyme. Lysine methyl ester as an analogue of the enzyme catalyzes the hydrolysis of NPA under similar experimental conditions. The results are described in terms of an assumed mechanism and the nature of the catalytic site is discussed.

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A study of the pH- and temperature-dependence of the reactions of yeast alcohol dehydrogenase with ethanol, acetaldehyde and butyraldehyde as substrates

Biochemical Journal ◽

10.1042/bj1470303 ◽

1975 ◽

Vol 147 (2) ◽

pp. 303-311 ◽

Cited By ~ 37

Author(s):

C J Dickenson ◽

F M Dickinson

Keyword(s):

Alcohol Dehydrogenase ◽

Temperature Dependence ◽

Initial Rate ◽

British Library ◽

Yeast Alcohol Dehydrogenase ◽

Wide Range ◽

Mechanism Of Catalysis ◽

Ph Range ◽

Kinetics Of ◽

Kinetics Of Reduction

The kinetics of ethanol oxidation by NAD+, and acetaldehyde and butyraldehyde reduction by NADH, catalysed by yeast alcohol dehydrogenase, were studied in the pH range 4.9--9.9 at 25 degrees C and in the temperature range 14.8--43.5 degrees C at pH 7.05. The kinetics of reduction of acetaldehyde by [4A-2H]NADH at pH 7.05 and pH 8.9 at 25 degrees C were also studied. The results of the kinetic experiments indicate that the mechanism of catalysis, previously proposed on the basis of studies at pH 7.05 and 25 degrees C (Dickinson & Monger, 1973), applies over the wide range of conditions now tested. Values of some of the initial-rate parameters obtained were used to deduce information about the pH- and temperature-dependence of the specific rates of combination of enzyme and coenzymes and of the dissociation of the enzyme--coenzyme compounds. Primary and secondary plots of initial-rate data are deposited as Supplementary Publication SUP 50043 (20 pages) with the British Library (Lending Division), Boston Spa, Wetherby, Yorks. LS23 7BQ, U.K., from whom copies may be obtained under the terms indicated in Biochem. J. (1975) 145, 5.

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Constant pH Molecular Dynamics Reveals How Proton Release Drives the Conformational Transition of a Transmembrane Efflux Pump

10.26434/chemrxiv.5496907 ◽

2017 ◽

Author(s):

Jana Shen ◽

Zhi Yue ◽

Helen Zgurskaya ◽

Wei Chen

Keyword(s):

Molecular Dynamics ◽

Conformational Changes ◽

Transmembrane Domain ◽

Conformational Transition ◽

Conformational Dynamics ◽

Proton Release ◽

Intrinsic Resistance ◽

Constant Ph ◽

Wide Range ◽

Constant Ph Molecular Dynamics

AcrB is the inner-membrane transporter of E. coli AcrAB-TolC tripartite efflux complex, which plays a major role in the intrinsic resistance to clinically important antibiotics. AcrB pumps a wide range of toxic substrates by utilizing the proton gradient between periplasm and cytoplasm. Crystal structures of AcrB revealed three distinct conformational states of the transport cycle, substrate access, binding and extrusion, or loose (L), tight (T) and open (O) states. However, the specific residue(s) responsible for proton binding/release and the mechanism of proton-coupled conformational cycling remain controversial. Here we use the newly developed membrane hybrid-solvent continuous constant pH molecular dynamics technique to explore the protonation states and conformational dynamics of the transmembrane domain of AcrB. Simulations show that both Asp407 and Asp408 are deprotonated in the L/T states, while only Asp408 is protonated in the O state. Remarkably, release of a proton from Asp408 in the O state results in large conformational changes, such as the lateral and vertical movement of transmembrane helices as well as the salt-bridge formation between Asp408 and Lys940 and other sidechain rearrangements among essential residues.Consistent with the crystallographic differences between the O and L protomers, simulations offer dynamic details of how proton release drives the O-to-L transition in AcrB and address the controversy regarding the proton/drug stoichiometry. This work offers a significant step towards characterizing the complete cycle of proton-coupled drug transport in AcrB and further validates the membrane hybrid-solvent CpHMD technique for studies of proton-coupled transmembrane proteins which are currently poorly understood. <p><br></p>

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Stopped-flow study of salt effects on the hydroxide and borate ion catalyzed hydrolysis of covalent p-tolylsulfonylmethyl perchlorate in aqueous borax buffer solutions

The Journal of Organic Chemistry ◽

10.1021/jo00412a051 ◽

1978 ◽

Vol 43 (18) ◽

pp. 3618-3621 ◽

Cited By ~ 5

Author(s):

Willem Breemhaar ◽

Jan B. F. N. Engberts

Keyword(s):

Stopped Flow ◽

Salt Effects ◽

Buffer Solutions ◽

Hydrolysis Of ◽

Flow Study

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Interactions of tervalent lanthanide ions with bacterial collagenase (clostridiopeptidase A)

Biochemical Journal ◽

10.1042/bj1950677 ◽

1981 ◽

Vol 195 (3) ◽

pp. 677-684 ◽

Cited By ~ 15

Author(s):

Christopher H. Evans

Keyword(s):

Ionic Radius ◽

Substrate Inhibition ◽

Volume Ratio ◽

Binding Pocket ◽

Lanthanide Ions ◽

Ionic Radii ◽

Apparent Dissociation Constant ◽

Substrate Complex ◽

Bacterial Collagenase ◽

Hydrolysis Of

Tervalent cations of the lanthanide (rare-earth) elements reversibly inhibit bacterial collagenase (clostridiopeptidase A; EC 3.4.24.3). Sm3+, whose ionic radius is closest to that of Ca2+, is the most effective inhibitor, completely suppressing clostridiopeptidase activity at a concentration of 100μm in the presence of 5mm-Ca2+. Er3+ and Lu3+, which both have ionic radii smaller than either Ca2+ or Sm3+, inhibit less efficiently, and La3+, which is slightly larger than Ca2+ or Sm3+, inhibits only weakly. These findings indicate a closely fitting, stereospecific, Ca2+-binding pocket in clostridiopeptidase, which excludes ions that are only slightly larger than Ca2+ [ionic radius 0.099nm (0.99 Ȧ)]. By contrast, trypsin, an enzyme whose activity does not depend on Ca2+, requires lanthanide concentrations 50–100-fold greater for inhibition. Furthermore, the relative efficiency of inhibition of trypsin by lanthanides increases as the lanthanide ions become smaller and the charge/volume ratio increases. At a concentration of 50μm, Sm3+ lowers the apparent Km for the hydrolysis of Pz-peptide by clostridiopeptidase from 5.4mm to 0.37mm and the apparent Vmax. from 0.29 Wünsch–Heidrich unit to 0.018 unit. Thus Sm3+ enhances the affinity of this enzyme for its substrate; inhibition of hydrolysis of Pz-peptide may result from the excessive stability of the enzyme–Sm3+–substrate complex. Inhibition by Sm3+ is competitive with regard to Ca2+. The apparent dissociation constant, Kd, of Ca2+ is 0.27mm, where the Ki for Sm3+ is 12μm. Clostridiopeptidase is more thermolabile in the absence of Ca2+. With Sm3+, thermoinactivation of the enzyme at 53°C or 60°C is initially accelerated, but then becomes retarded as heating continues. Lanthanide ions bind to gelatin and collagen. In so doing, they appear to protect these substrates from lysis by clostridiopeptidase through mechanisms additional to supplanting Ca2+ at its binding site on the enzyme. Collagen and gelatin sequester sufficient lanthanide ions to gain partial protection from clostridiopeptidase in the absence of an extraneous source of these inhibitors.

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Phosphatase synthesis in Klebsiella (Aerobacter) aerogenes growing in continuous culture

Biochemical Journal ◽

10.1042/bj1270087 ◽

1972 ◽

Vol 127 (1) ◽

pp. 87-96 ◽

Cited By ~ 28

Author(s):

P. G. Bolton ◽

A. C. R. Dean

Keyword(s):

Alkaline Phosphatase ◽

Acid Phosphatase ◽

Continuous Culture ◽

Activity Profile ◽

Glucose Effect ◽

Ph Values ◽

Nitrophenyl Phosphate ◽

Wide Range ◽

Rate Of Hydrolysis ◽

Hydrolysis Of

1. Phosphatase synthesis was studied in Klebsiella aerogenes grown in a wide range of continuous-culture systems. 2. Maximum acid phosphatase synthesis was associated with nutrient-limited, particularly carbohydrate-limited, growth at a relatively low rate, glucose-limited cells exhibiting the highest activity. Compared with glucose as the carbon-limiting growth material, other sugars not only altered the activity but also changed the pH–activity profile of the enzyme(s). 3. The affinity of the acid phosphatase in glucose-limited cells towards p-nitrophenyl phosphate (Km 0.25–0.43mm) was similar to that of staphylococcal acid phosphatase but was ten times greater than that of the Escherichia coli enzyme. 4. PO43−-limitation derepressed alkaline phosphatase synthesis but the amounts of activity were largely independent of the carbon source used for growth. 5. The enzymes were further differentiated by the effect of adding inhibitors (F−, PO43−) and sugars to the reaction mixture during the assays. In particular, it was shown that adding glucose, but not other sugars, stimulated the rate of hydrolysis of p-nitrophenyl phosphate by the acid phosphatase in carbohydrate-limited cells at low pH values (<4.6) but inhibited it at high pH values (>4.6). Alkaline phosphatase activity was unaffected. 6. The function of phosphatases in general is discussed and possible mechanisms for the glucose effect are outlined.

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The mechanism of the oxidation of thiocyanate ion by peroxomonosulphate in aqueous solution. II. Kinetics of the reaction

Australian Journal of Chemistry ◽

10.1071/ch9661365 ◽

1966 ◽

Vol 19 (8) ◽

pp. 1365 ◽

Cited By ~ 4

Author(s):

RH Smith ◽

IR Wilson

Keyword(s):

Aqueous Solution ◽

Initial Rate ◽

Stopped Flow ◽

Thiocyanate Ion ◽

First Order ◽

Conductance Method ◽

Rate Of Reaction ◽

Kinetics Of

Initial rates of reaction for the above oxidation have been measured by a stopped-flow conductance method. Between pH 2 and 3.6, the initial rate of reaction, R, is given by the expression R{[HSO5-]+[SCN-]} = {kb+kc[H+]}[HSO5-]0[SCN-]20+ka[H+]-1[HSO5]20[SCN-]0 As pH increases, there is a transition to a pH-independent rate, first order in each thiocyanate and peroxomonosulphate concentrations.

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