The Observation of Acyl-Enzyme Intermediates in the α-Chymotrypsin-Catalyzed Hydrolysis of Specific Ester Substrates at Low pH

1964 ◽  
Vol 86 (5) ◽  
pp. 937-938 ◽  
Author(s):  
Ferenc J. Kezdy ◽  
Myron L. Bender
Keyword(s):  
Low Ph ◽  
Enzyme Intermediates ◽  
Hydrolysis Of

scholarly journals Dissociation of clathrin coats coupled to the hydrolysis of ATP: role of an uncoating ATPase.

1984 ◽  
Vol 99 (2) ◽  
pp. 734-741 ◽  
Author(s):  
W A Braell ◽  
D M Schlossman ◽  
S L Schmid ◽  
J E Rothman
Keyword(s):  
Atpase Activity ◽  
Atp Hydrolysis ◽  
Low Ph ◽  
Coated Vesicles ◽  
Magnesium Concentration ◽  
Cross Linking ◽  
Dependent Atpase ◽  
Event Triggering ◽  
Hydrolysis Of

ATP hydrolysis was used to power the enzymatic release of clathrin from coated vesicles. The 70,000-mol-wt protein, purified on the basis of its ATP-dependent ability to disassemble clathrin cages, was found to possess a clathrin-dependent ATPase activity. Hydrolysis was specific for ATP; neither dATP nor other ribonucleotide triphosphates would either substitute for ATP or inhibit the hydrolysis of ATP in the presence of clathrin cages. The ATPase activity is elicited by clathrin in the form of assembled cages, but not by clathrin trimers, the product of cage disassembly. The 70,000-mol-wt polypeptide, but not clathrin, was labeled by ATP in photochemical cross-linking, indicating that the hydrolytic site for ATP resides on the uncoating protein. Conditions of low pH or high magnesium concentration uncouple ATP hydrolysis from clathrin release, as ATP is hydrolyzed although essentially no clathrin is released. This suggests that the recognition event triggering clathrin-dependent ATP hydrolysis occurs in the absence of clathrin release, and presumably precedes such release.

scholarly journals Chemical synthesis and papain-catalysed hydrolysis of N-α-benzyloxycarbonyl-l-lysine p-nitroanilide

1985 ◽  
Vol 226 (2) ◽  
pp. 601-606 ◽  
Author(s):  
N E Mackenzie ◽  
J P G Malthouse ◽  
A I Scott
Keyword(s):  
Chemical Synthesis ◽  
Ph Dependence ◽  
Low Ph ◽  
Pka Values ◽  
Tetrahedral Intermediate ◽  
Ph Values ◽  
Km Value ◽  
Hydrolysis Of

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.

scholarly journals Identification of Nucleophilic Probes for Protease-Mediated Transpeptidation

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2109 ◽  
Author(s):  
Ga-eul Eom ◽  
Seokhee Kim
Keyword(s):  
Mild Condition ◽  
Peptide Bonds ◽  
Selective Enrichment ◽  
Screening Approach ◽  
Enzyme Intermediates ◽  
Covalently Linked ◽  
Hydrolysis Of ◽  
Enzyme Intermediate

Proteases have evolved to mediate the hydrolysis of peptide bonds but may perform transpeptidation in the presence of a proper nucleophilic molecule that can effectively compete with water to react with the acyl-enzyme intermediate. There have been several examples of protease-mediated transpeptidation, but they are generally inefficient, and little effort has been made to systematically control the transpeptidation activity of other proteases with good nucleophiles. Here, we developed an on-bead screening approach to find a probe that functions efficiently as a nucleophile in the protease-mediated transpeptidation reaction, and we identified good probes for a model protease DegP. These probes were covalently linked to the C-termini of the cleaved peptides in a mild condition and made the selective enrichment of ligated peptides possible. We suggest that good nucleophilic probes can be found for many other proteases that act via acyl-enzyme intermediates, and these probes will help characterize their substrates.

scholarly journals Stopped-Flow Kinetic Study of Reduction of Ferric Maltol Complex by Ascorbate

2016 ◽  
Vol 12 (4) ◽  
pp. 4338-4341
Author(s):  
Shabana Amin ◽  
Shazia Nisar ◽  
S Arif Kazmi
Keyword(s):  
Kinetic Study ◽  
Acid Hydrolysis ◽  
Rate Constant ◽  
Free Ligand ◽  
Low Ph ◽  
Reducing Agent ◽  
The Other ◽  
Stopped Flow ◽  
Kinetic Investigation ◽  
Hydrolysis Of

Stopped-flow kinetic investigation of reduction of Fe(III)-maltol complex is reported. The rates are dependent on pH in a complex way. On one hand at low pH there is a predominance of Fe(III)(maltol)2 which is easier to reduce compared to Fe(III) (maltol)3 which is more resistant to reduction. On the other hand ascorbate is a stronger reducing agent at higher pH. The rates are also found to be inversely dependent on the concentration of free ligand. These observations are explained by the following rate law:Rate = ((k0 +k1[H+])k2 [Asc-]/ (k-1[HMal] + k2[Asc-])) + k3 [Asc-] ) [FeIII(Mal)3] Here k1 is the rate constant for acid hydrolysis of the Fe(maltol)3 complex to Fe(maltol)2 complex and is directly controlled by H+, k0 is the rate constant for hydrolysis of the Fe(maltol)3 complex to Fe(maltol)2 complex and is an intrinsic process, k-1 is the rate constant of reformation of the tris complex by reaction of the bis complex and the free ligand, k2 is the rate constant for reduction of the bis complex by ascorbate and k3 is the rate constant for the reduction of the tris complex by ascorbate.

A change in rate-determining step in the hydrolysis of cyclic ketals

1984 ◽  
Vol 62 (8) ◽  
pp. 1608-1612
Author(s):  
Robert A. McClelland ◽  
N. Esther Seaman
Keyword(s):  
Bond Cleavage ◽  
Structural Features ◽  
Product Formation ◽  
Low Ph ◽  
Alkoxy Group ◽  
Hydroxide Ion ◽  
Rate Determining Step ◽  
The Difference ◽  
Ortho Esters ◽  
Hydrolysis Of

A kinetic study is reported of the hydrolysis of 2-methoxy-2-phenyltetrahydrofuran and 2-ethoxy-2-phenyltetrahydrofuran. At pH > 6 the rate-determining step involves H+-catalyzed formation of the oxocarbocation, this reaction occurring with cleavage of the exocyclic alkoxy group to produce a cyclic cation. Between pH 5 and pH 6 a change-over occurs and at pH < 5, the rate-determining step in product formation is breakdown of the cyclic hemiketal intermediate, 2-hydroxy-2-phenyltetrahydrofuran. The changeover occurs because the H+-catalyzed breakdown of this intermediate is a slower process than the H+-catalyzed oxocarbocation-forming step. Hydroxide ion catalysis makes the hemiketal decomposition faster at higher pH. Analogous cyclic ortho esters (2-alkoxy-1,3-dioxolanes) show this same change in rate-determining step between high pH and low pH, while acyclic acetals, ketals, and ortho esters generally have the oxocarbocation-forming stage rate determining at all acidities. It is concluded that the structural features inherent in the cyclic systems are responsible for the difference. In particular, the oxocarbocation-forming stage involves exocyclic bond cleavage, giving it an entropic advantage over the hemiketal or hemiorthoester breakdown which is endocyclic.

scholarly journals The Influence of pH on the Combustion Properties of Bio-Coal Following Hydrothermal Treatment of Swine Manure

Energies ◽  
2020 ◽  
Vol 13 (2) ◽  
pp. 331 ◽  
Author(s):  
Aidan Mark Smith ◽  
Ugochinyere Ekpo ◽  
Andrew Barry Ross
Keyword(s):  
Energy Content ◽  
Swine Manure ◽  
Low Ph ◽  
Mineral Matter ◽  
Fuel Processing ◽  
Combustion Properties ◽  
Higher Temperature ◽  
Decreasing Ph ◽  
Influence Of Ph ◽  
Hydrolysis Of

The application of excessive amounts of manure to soil prompted interest in using alternative approaches for treating slurry. One promising technology is hydrothermal carbonisation (HTC) which can recover nutrients such as phosphorus and nitrogen while simultaneously making a solid fuel. Processing manure under acidic conditions can facilitate nutrient recovery; however, very few studies considered the implications of operating at low pH on the combustion properties of the resulting bio-coal. In this work, swine manure was hydrothermally treated at temperatures ranging from 120 to 250 °C in either water alone or reagents including 0.1 M NaOH, 0.1 M H2SO4, and finally 0.1 M organic acid (CH3COOH and HCOOH). The influence of pH on the HTC process and the combustion properties of the resulting bio-coals was assessed. The results indicate that pH has a strong influence on ash chemistry, with decreasing pH resulting in an increased removal of ash. The reduction in mineral matter influences the volatile content of the bio-coal and its energy content. As the ash content in the final bio-coal reduces, the energy density increases. Treatment at 250 °C results in a more “coal like” bio-coal with fuel properties similar to that of lignite coal and a higher heating value (HHV) ranging between 21 and 23 MJ/kg depending on pH. Processing at low pH results in favourable ash chemistry in terms of slagging and fouling. Operating at low pH also appears to influence the level of dehydration during HTC. The level of dehydration increases with decreasing pH, although this effect is reduced at higher temperatures. At higher-temperature processing (250 °C), operating at lower pH increases the yield of bio-coal; however, at lower temperatures (below 200 °C), the reverse is true. The lower yields obtained below 200 °C in the presence of acid may be due to acid hydrolysis of carbohydrate in the manure, whereas, at the higher temperatures, it may be due to the acid promoting polymerisation.

Hydrolysis of trioxaadamantane ortho esters. II. Kinetic analysis and the nature of the rate-determining step

1984 ◽  
Vol 62 (6) ◽  
pp. 1074-1080 ◽  
Author(s):  
Robert A. McClelland ◽  
Patrick W. K. Lam
Keyword(s):  
Rate Constants ◽  
Product Formation ◽  
Low Ph ◽  
Acid Solutions ◽  
Ortho Ester ◽  
Rate Determining Step ◽  
Acid Catalyzed ◽  
Cation Hydration ◽  
Ortho Esters ◽  
Hydrolysis Of

A detailed kinetic study of the hydrolysis of a series of 3-aryl-2,4,10-trioxaadamantanes is reported. These ortho esters equilibrate with the ring-opened dialkoxycarbocation, in a very rapid process which could be studied using temperature-jump spectroscopy for aryl = 2,4-dimethylphenyl. Relaxation rate constants are of the order of 104 s−1; these could be analyzed to provide the rate constants for both the ring opening and the ring closing. Product formation from this equilibrating mixture is much slower. In acid solutions (0.01 M H+ −50% H2SO4), first-order rate constants for product formation initially increase with increasing acidity, but a maximum is reached at 20–35% H2SO4 and the rate then falls. This behavior is explained by a counterbalancing of two factors. Increasing acidity increases the amount of the dialkoxycarbocation in the initial equilibrium, but, outside the pH region, it decreases the rate of hydrolysis of this cation through a medium effect. Rate constants over a range of pH have been measured for two trioxaadamantanes and for the cation DEt+ derived by treatment of the ortho ester with triethyloxonium tetraafluoroborate. The latter models the cation formed in the ortho ester hydrolysis but it cannot ring close. Rate–pH profiles obtained in these systems are more complex than expected on the basis of rate-determining cation hydration. An interpretation is proposed with a change in rate-determining step between high pH and low pH. Cation hydration is rate determining at high pH but at low pH hemiorthoester decomposition becomes rate determining. Under these conditions the hemiorthoester equilibrates with both the dialkoxycarbocation and with the trioxaadamantane. The change in rate-determining step occurs because acid-catalyzed reversion of the hemiorthoester to dialkoxycarbocation is a faster process than acid-catalyzed hemiorthoester decomposition. This makes the latter rate-determining in acid solutions. Additional pathways available to the decomposition, however, make it the faster process at higher pH. A kinetic analysis furnishes all of the rate and equilibrium constants for the system, and provides support for the mechanistic interpretation. A comparison of these numbers with those obtained for the three stages in the hydrolysis of a simple monocyclic ortho ester underlines the novelty of the trioxaadamantane system.

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