Hydrolysis of N-acetyl-p-benzoquinone imines: pH dependence of the partitioning of a tetrahedral intermediate

1989 ◽  
Vol 111 (12) ◽  
pp. 4447-4456 ◽  
Author(s):  
Michael Novak ◽  
Gayl A. Bonham ◽  
Julio J. Mulero ◽  
Maria Pelecanou ◽  
Joseph N. Zemis ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Tetrahedral Intermediate ◽  
Hydrolysis Of

ChemInform Abstract: Hydrolysis of N-Acetyl-p-benzoquinone Imines: pH Dependence of the Partitioning of a Tetrahedral Intermediate

ChemInform ◽  
1989 ◽  
Vol 20 (39) ◽  
Author(s):  
M. NOVAK ◽  
G. A. BONHAM ◽  
J. J. MULERO ◽  
M. PELECANOU ◽  
J. N. ZEMIS ◽  
...  
Keyword(s):  
Ph Dependence ◽  
Tetrahedral Intermediate ◽  
Hydrolysis Of

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.

pH Effects in trypsin catalysis

1969 ◽  
Vol 47 (21) ◽  
pp. 4021-4029 ◽  
Author(s):  
H. P. Kasserra ◽  
K. J. Laidler
Keyword(s):  
Complex Formation ◽  
Ph Dependence ◽  
Specific Rotation ◽  
Ph Effects ◽  
Ph Values ◽  
A Value ◽  
Available Information ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Covalent Complex

A kinetic study has been made of the trypsin-catalyzed hydrolysis of N-benzoyl-L-alanine methyl ester, at pH values ranging from 6 to 10. The substrate concentrations varied from 1.7 × 10−3 to 4.3 × 10−2 M. From the rates were calculated, at each pH, values of [Formula: see text] (corresponding to [Formula: see text]), [Formula: see text] (corresponding to [Formula: see text]) and [Formula: see text] The specific levorotation of trypsin was measured and found to vary with pH in the pH region 5–11, the change in specific rotation following the ionization of a single group with pK(app) of 9.4. At pH 11 the specific rotation of trypsin, its zymogen, and its phosphorylated derivative were approximately the same, suggesting similar conformations for all three forms of the protein.The kinetic results on the acid side were very similar to those obtained by other investigators for chymotrypsin; they imply that there is a group of [Formula: see text] in the free enzyme, presumably the imidazole function of a histidine residue, and that this group is involved in acylation and deacylation, which can only occur if it is unprotonated. The behavior on the basic side was found to be different from that with chymotrypsin revealing a decrease in [Formula: see text] at high pH corresponding to a value of [Formula: see text] whereas [Formula: see text] showed sigmoid pH-dependence. An interpretation of these results that is consistent with all available information is that a group of [Formula: see text] (presumably the —NH3+ function of the terminal isoleucine) controls the conformation and thereby the activity of the enzyme at different stages of complex formation. In contrast to chymotrypsin, the pK of this ionizing group appears to be generally lowered by covalent complex formation between trypsin and its substrates.

Covalently Bound Flavin in D-6-Hydroxynicotine Oxidase from Arthrobacter oxidans

1972 ◽  
Vol 27 (9) ◽  
pp. 1073-1074 ◽  
Author(s):  
M. Brühmüller ◽  
H. Möhler ◽  
K. Decker
Keyword(s):  
Amino Acid ◽  
Flavin Adenine Dinucleotide ◽  
Ph Dependence ◽  
Spectral Characteristics ◽  
Amino Acid Derivative ◽  
Aminopeptidase M ◽  
Arthrobacter Oxidans ◽  
Layer Chromatography ◽  
Hydrolysis Of ◽  
Covalently Bound

D-6-hydroxynicotine oxidase contains 1 mole of FAD covalently bound to one mole of enzyme. To identify the covalent linkage between FAD and protein, an amino acid derivative of riboflavin (HNO-flavin) was isolated and purified. It was obtained from flavin peptides by hydrolysis with 6 N HCl at 95°C or with aminopeptidase M. The riboflavin derivative had the spectral characteristics of 8α-substituted flavins. It showed a pH-dependence of fluorescence with a pK of 4.65 and 86% quenching at pH 7. In thin layer chromatography it was identical with 8α-(N-3-histidyl)-riboflavin. Hydrolysis of HNO-flavin in 6 N HCl at 125°C liberated 1 mole of histidine per mole of flavin as shown by amino acid analysis. Since FAD is the coenzyme of D-6-hydroxynicotine oxidase, these results are taken as evidence that this enzyme contains 8a- (N-3-histidyl) -flavin-adenine-dinucleotide in the active center.

scholarly journals Cathepsin B, the lysosomal thiol proteinase of calf liver

1969 ◽  
Vol 114 (4) ◽  
pp. 673-678 ◽  
Author(s):  
O. Snellman
Keyword(s):  
Cathepsin B ◽  
Ph Dependence ◽  
Gel Filtration ◽  
Chelating Agent ◽  
Thiol Group ◽  
Maximum Activity ◽  
Active Centre ◽  
Peptide Bonds ◽  
Hydrolysis Of ◽  
Thiol Proteinase

Cathepsin B from calf liver was obtained by a method involving preparation of a lysosomal–mitochondrial pellet and treatment of this pellet with acetone. The material was extracted with an acid buffer, pH4·0, and then precipitated from the extract with acetone. The precipitate was dissolved in phosphate buffer, pH7·4, and subjected to gel filtration on Sephadex G-200 and G-100. The cathepsin B emerged in a range of molecular weight much lower than 50000 as a well-defined component. The purity of this material was checked by electrophoresis. To obtain maximum activity the enzyme had to be activated with a chelating agent and a reducing agent (i.e. EDTA and cysteine). A number of different substrates were used. The enzyme was active for the hydrolysis of both peptide bonds and ester bonds and had approximately equal reactivity in the two cases. The pH-dependence of the hydrolysis was the same with both substrates. The binding of the substrates was half-maximal at pH4·5 and at pH6·8. A thiol group occurred in the active centre but this group ought to have a much higher pK than that found in this enzyme.

A comparison of the mechanisms of hydrolysis of benzimidates, esters, and amides in sulfuric acid media

2005 ◽  
Vol 83 (9) ◽  
pp. 1391-1399 ◽  
Author(s):  
Robin A Cox
Keyword(s):  
Sulfuric Acid ◽  
Isotope Effects ◽  
Ester Hydrolysis ◽  
Water Molecules ◽  
Acid Media ◽  
Tetrahedral Intermediate ◽  
Amide Hydrolysis ◽  
Solvent Isotope ◽  
Reaction Media ◽  
Hydrolysis Of

The mechanisms given in textbooks for both ester and amide hydrolysis in acid media are in need of revision. To illustrate this, benzimidates were chosen as model compounds for oxygen protonated benzamides. In aqueous sulfuric acid media they hydrolyze either by a mechanism involving attack of two water molecules at the carbonyl carbon to give a neutral tetrahedral intermediate directly, as in ester hydrolysis, or by an SN2 attack of two water molecules at the alkyl group of the alkoxy oxygen to form the corresponding amide, or by both mechanisms, depending on the structure of the benzimidate. The major line of evidence leading to these conclusions is the behavior of the excess acidity plots resulting from the rate constants obtained for the hydrolyses as functions of acid concentration and temperature. The first of these mechanisms is in fact very similar to one found for the hydrolysis of benzamides, as inferred from: (1) similar excess acidity plot behaviour; and (2) the observed solvent isotope effects for amide hydrolysis, which are fully consistent with the involvement of two water molecules, but not with one or with three (or more). This mechanism starts out as essentially the same one as that found for ester hydrolysis under the same conditions. Differences arise because the neutral tetrahedral intermediate, formed directly as a result of the protonated substrate being attacked by two water molecules (not one), possesses an easily protonated nitrogen in the amide and benzimidate cases, explaining both the lack of 18O exchange observed for amide hydrolysis and the irreversibility of the reaction. Protonated tetrahedral intermediates are too unstable to exist in the reaction media; in fact, protonation of an sp3 hybridized oxygen to put a full positive charge on it is extremely difficult. (This means that individual protonated alcohol or ether species are unlikely to exist in these media either.) Thus, the reaction of the intermediate going to product or exchanged reactant is a general-acid-catalyzed process for esters. For amide hydrolysis, the situation is complicated by the fact that another, different, mechanism takes over in more strongly acidic media, according to the excess acidity plots. Some possibilities for this are given.Key words: esters, amides, benzimidates, hydrolysis, excess acidity, mechanism, acid media.

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