scholarly journals Human cathepsin B. Application of the substrate N-benzyloxycarbonyl-l-arginyl-l-arginine 2-naphthylamide to a study of the inhibition by leupeptin

1980 ◽  
Vol 189 (3) ◽  
pp. 447-453 ◽  
Author(s):  
C G Knight
Keyword(s):  
Kinetic Parameters ◽  
Cathepsin B ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Mean Value ◽  
Efficient Synthesis ◽  
The Mean ◽  
Human Cathepsin ◽  
Ph Range ◽  
Hydrolysis Of

1. The kinetic parameters Kcat. and Km were determined for the hydrolysis of some arginine naphthylamides by human cathepsin B. 2. A new and efficient synthesis of Z-Arg-Arg-NNap (benzyloxycarbonyl-L-arginyl-L-arginine 2-naphthylamide) was developed. 3. Z-Arg-Arg-NNap was a specific and sensitive substrate for cathepsin B, and was used for kinetic studies. 4. Values of kcat. were maximal in the pH range 5.4–6.2, and depended on a single ionizing group of pKa 4.4. 5. Leupeptin was a purely competitive inhibitor of human cathepsin B. 6. The effect of pH on the apparent inhibitor constant, Ki (app.), was determined. Ki (app.) was pH-independent in the range pH 4.3–6.0, with the mean value 7 × 10(-9) M.

Micellar catalysis of organic reactions. VIII. Kinetic studies of the hydrolysis of 2-acetyloxybenzoic acid (aspirin) in the presence of micelles

1982 ◽  
Vol 35 (7) ◽  
pp. 1357 ◽  
Author(s):  
TJ Broxton
Keyword(s):  
Aqueous Solution ◽  
Cetyltrimethylammonium Bromide ◽  
Cetylpyridinium Chloride ◽  
Kinetic Studies ◽  
Base Catalysis ◽  
Plateau Region ◽  
General Base ◽  
Mechanism Of Hydrolysis ◽  
Ph Range ◽  
Hydrolysis Of

The hydrolysis of 2-acetyloxybenzoic acid in the pH range 6-12 has been studied in the presence of micelles of cetyltrimethylammonium bromide (ctab) and cetylpyridinium chloride (cpc). In the plateau region (pH 6-8) the hydrolysis is inhibited by the presence of micelles, while in the region where the normal BAC2 hydrolysis (pH > 9) occurs the reaction is catalysed by micelles of ctab and cpc. The mechanism of hydrolysis in the plateau region is shown to involve general base catalysis by the adjacent ionized carboxy group both in the presence and absence of micelles. This reaction is inhibited in the presence of micelles because the substrate molecules are solubilized into the micelle and water is less available in this environment than in normal aqueous solution.

scholarly journals Kinetic studies on the acid hydrolysis of the methyl ketoside of unsubstituted and O-acetylated N-acetylneuraminic acid

1973 ◽  
Vol 133 (4) ◽  
pp. 623-628 ◽  
Author(s):  
A. Neuberger ◽  
Wendy A. Ratcliffe
Keyword(s):  
Sialic Acid ◽  
Acid Hydrolysis ◽  
Model Compound ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Neuraminic Acid ◽  
Acetylneuraminic Acid ◽  
Rate Of Hydrolysis ◽  
Ph Range ◽  
Hydrolysis Of

The hydrolysis of the model compound 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl-α-d-neuraminic acid and neuraminidase (Vibrio cholerae) closely resembled that of the O-acetylated sialic acid residues of rabbit Tamm–Horsfall glycoprotein. This confirmed that O-acetylation was responsible for the unusually slow rate of acid hydrolysis of O-acetylated sialic acid residues observed in rabbit Tamm–Horsfall glycoprotein and their resistance to hydrolysis by neuraminidase. The first-order rate constant of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid by 0.05m-H2SO4 was 56-fold greater than that of 2-O-methyl-4,7,8,9-tetra-O-acetyl-N-acetyl -α-d-neuraminic acid. Kinetic studies have shown that in the pH range 1.00–3.30, the observed rate of hydrolysis of 2-methyl-N-acetyl-α-d-neuraminic acid can be attributed to acid-catalysed hydrolysis of the negatively charged CO2− form of the methyl ketoside.

Kinetic studies of modifier effects on the carboxypeptidase A catalyzed hydrolyses of peptides

1987 ◽  
Vol 65 (8) ◽  
pp. 717-725 ◽  
Author(s):  
John F. Sebastian ◽  
Richard S. Hinks ◽  
Ralf V. Reuland
Keyword(s):  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Peptidase Activity ◽  
Benzene Sulfonate ◽  
Carboxypeptidase A ◽  
Structural Requirements ◽  
Detailed Mechanism ◽  
Hydrolysis Of ◽  
Phenyl Alanine ◽  
Standing Question

A variety of modifiers of carboxypeptidase A (CPA) have been investigated in an effort to understand the structural requirements of inhibitors and activators of peptidase activity. It is proposed that an understanding of the mechanism of action of reversible activators of the enzyme may bear on the long standing question of whether the detailed mechanism of peptidase activity is different from that of esterase activity. An analog of the activator 2,2-dimethyl-2-silapentane-5-sulfonate, 5,5-dimethylhexanoate, was found to be a competitive inhibitor of the CPA-catalyzed hydrolysis of benzoylglycyl-L-phenyl-alanine. The modifier 4-phenyl-3-butenoate (styrylacetic acid) was determined to be an activator. The sulfonates benzene-sulfonate, p-toluenesulfonate, phenylmethanesulfonate, 2-phenylethanesulfonate, and 3-phenylpropanesulfonate were all found to be activators.

A fluorogenic substrate for angiotensin-converting enzyme in plasma.

1978 ◽  
Vol 24 (9) ◽  
pp. 1539-1542 ◽  
Author(s):  
F S Russo ◽  
A V Persson ◽  
I B Wilson
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Human Plasma ◽  
Mean Value ◽  
Converting Enzyme ◽  
Fluorogenic Substrate ◽  
Fluorescent Substrate ◽  
The Mean ◽  
Hydrolysis Of ◽  
Assay Conditions

Abstract A simple, sensitive, and reproducible assay for angiotensin-converting enzyme is described. It is based on the hydrolysis of the minimally fluorescent substrate p-nitrobenzyloxycarbonylglycyl-L-tryptophylglycine to the products p-nitrobenzyloxycarbonylglycine and the highly fluorescent L-tryptophylglycine. The L-tryptophylglycine was analyzed by fluorometry (lambda excitation = 285 nm; lambda emission = 350 nm). The mean value for human plasma (serum) is 16.5 nmol of substrate hydrolyzed per minute per milliter of plasma under the described assay conditions.

Aspects of the hydrolysis of formamide: revisitation of the water reaction and determination of the solvent deuterium kinetic isotope effect in base

2002 ◽  
Vol 80 (10) ◽  
pp. 1343-1350 ◽  
Author(s):  
H Slebocka-Tilk ◽  
F Sauriol ◽  
Martine Monette ◽  
R S Brown
Keyword(s):  
Isotope Effect ◽  
Kinetic Isotope Effect ◽  
Activation Parameters ◽  
Kinetic Studies ◽  
Base Hydrolysis ◽  
Kinetic Isotope ◽  
Acid And Base ◽  
Acid Reaction ◽  
Ph Range ◽  
Hydrolysis Of

A study of the hydrolysis of formamide is reported with the aims of isolating the water reaction for hydrolysis from the acid and base hydrolysis terms and determining the solvent deuterium kinetic isotope effect (dkie) on base-catalyzed hydrolysis. Respective activation parameters (ΔH‡ and ΔS‡) of (17.0 ± 0.4) kcal mol–1 and (–18.8 ± 1.3) cal mol–1 K–1 for the acid reaction and (17.9 ± 0.2) kcal mol–1 and (–11.1 ± 0.5) cal mol–1 K–1 for the base reaction were determined from Eyring plots of the second-order rate constants over the range of 27–120°C. Kinetic studies at the minima of the pH/rate profiles in the pH range from 5.6 to 6.2 in MES buffers at 56°C, and in the pH range of 4.25–6.87 in acetate and phosphate buffers at 120°C are reported. At 56°C the available data fit the expression k56obs = 0.00303[H3O+] + 0.032[HO–] + (3.6 ± 0.1) × 10–9, while at 120°C the data fit k120obs = (0.15 ± 0.02)[H3O+] + (3.20 ± 0.24)[HO–] + (1.09 ± 0.29) × 10–6. Preliminary experimental estimates of Ea (ln A) of 22.5 kcal mol–1 (15.03) for the water rate constant (kw) are calculated from an Arrhenius plot of the 56 and 120°C data giving an estimated kw of 1.1 × 10–10 s–1 (t1/2 = 199 years) at 25°C. Solvent dkie values of kOH/kOD = 1.15 and 0.77 ± 0.06 were determined at [OL–] = 0.075 and 1.47 M, respectively. The inverse value is determined under conditions where the the first step of the reaction dominates and is analyzed in terms of a rate-limiting attack of OL–.Key words: formamide, activation parameters, water reaction, acid and base hydrolysis, solvent kinetic isotope effect.

scholarly journals Rat small-intestinal β-galactosidases. Kinetic studies with three separated fractions

1968 ◽  
Vol 110 (1) ◽  
pp. 143-150 ◽  
Author(s):  
Nils-Georg Asp ◽  
Arne Dahlqvist
Keyword(s):  
Active Sites ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Small Intestinal Mucosa ◽  
Lactase Activity ◽  
Freezing And Thawing ◽  
Ph Optimum ◽  
Measurable Rate ◽  
Small Intestinal ◽  
Hydrolysis Of

1. Three fractions of β-galactosidase activity from the rat small-intestinal mucosa were separated chromatographically. Two of these fractions had an acid pH optimum at 3–4, and the third one had a more neutral pH optimum at 5·7. 2. The two ‘acid’ β-galactosidase fractions had considerably lower Km values for hetero β-galactosides than for lactose. The Vmax. values were similar for all the substrates used (lactose, phenyl β-galactoside, o-nitrophenyl β-galactoside, p-nitrophenyl β-galactoside and 6-bromo-2-naphthyl β-galactoside). No difference could be detected between the two ‘acid’ fractions with respect to their enzymic properties (pH optimum, Km for the different substrates, Ki for lactose as an inhibitor of the hydrolysis of hetero β-galactosides, Ki for phenyl β-galactoside as an inhibitor of the hydrolysis of lactose, and relative Vmax. for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ‘neutral’ fraction had similar Km values for all the substrates hydrolysed, but with lactose as substrate the Vmax. was much higher than with the hetero β-galactosides. This fraction did not split phenyl β-galactoside or 6-bromo-2-naphthyl β-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero β-galactosidase activities of all the three fractions, and Ki for lactose as an inhibitor in each case was the same as Km for the lactase activity. Phenyl β-galactoside was a competitive inhibitor of the lactase activity of all the three fractions. These facts strongly indicate that in all the three fractions lactose is hydrolysed by the same active sites as the hetero β-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.

The Steady State Kinetics of Plasmin and Trypsin Catalysed Hydrolysis of a Number of Tripeptideanilides

1979 ◽  
Author(s):  
U. Christensen ◽  
H-H. Ipsen
Keyword(s):  
Steady State ◽  
Kinetic Parameters ◽  
Amino Group ◽  
Steady State Kinetics ◽  
Steady State Kinetic ◽  
Ph Range ◽  
Kinetics Of ◽  
Hydrolysis Of ◽  
State Kinetic

The steady state kinetic parameters of plasmin and trypsin catalysed hydrolysis of Bz-L-Phe-Val-Arg-pNA, L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA, D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA in the pH-range 6-9 are presented. Ionization of catalytically essential enzymic groups accounts satisfactorily for the pH-dependencies of the kinetic parameters for plas-rain and trypsin reactions with Bz-L-Phe-Val-Arg-pNA, Bz-D-Phe-Val-Arg-pNA and D-Val-Leu-Lys-pNA. The protonation of the α-amino group of L-Phe-Val-Arg-pNA and D-Phe-Val-Arg-pNA (pK=7.0) show some additional effect. The values of the catalytic constants for plasmin and trypsin reactions with these p-nitroanilides are alike those normally found for specific ester substrates, indicating that the deacylation steps are rate determining.

HEMOLYTIC ASSAY OF THE NINTH COMPLEMENT COMPONENT: ELEVATION AND DEPLETION IN RHEUMATIC DISEASES

1971 ◽  
Vol 134 (3) ◽  
pp. 259-275 ◽  
Author(s):  
Shaun Ruddy ◽  
Lloyd K. Everson ◽  
Peter H. Schur ◽  
K. Frank Austen
Keyword(s):  
Renal Disease ◽  
Rheumatic Diseases ◽  
Fluid Phase ◽  
Kinetic Studies ◽  
Mean Value ◽  
Normal Serum ◽  
Normal Range ◽  
Hemolytic Assay ◽  
Normal Individuals ◽  
The Mean

An effective molecule titration for the ninth component of complement in the biologic fluids of man was developed using EAC1-8 cells produced by treating EAC14 cells with a chromatographic fraction of human serum containing C2, C3, C5, C6, C7, and C8. Kinetic studies of the interaction of EAC1-8 with C9 indicated that this component was depleted from the fluid phase, and that the lytic reaction proceeded most rapidly at ionic strength 0.145, and at a temperature of 37°C. The mean value for C9 in normal serum was 52,000 ±12,000 units/ml. The mean serum C9 for patients with DJD, rheumatoid arthritis, or SLE without active renal disease was approximately twice the mean for normal individuals. Patients with SLE and active renal disease had a mean C9 value which fell within the normal range, but was significantly lower than in patients with SLE who did not have active renal disease. Two instances of absolutely subnormal C9 levels were observed in patients during attacks of florid SLE, including nephritis. Since the usual change in serum C9 in rheumatic diseases is a marked elevation, the occurrence of a subnormal value reflects circumstances in which depletion due to activation of the sequence exceeds the increases associated with the inflammatory response.

Equilibrium and kinetic studies of the interaction of site-specific ligands with acetylcholinesterase from Electrophorus electricus

1978 ◽  
Vol 56 (12) ◽  
pp. 1133-1140 ◽  
Author(s):  
George Tomlinson ◽  
Bulent Mutus ◽  
W. John Rutherford
Keyword(s):  
Steady State ◽  
Dissociation Constant ◽  
Kinetic Studies ◽  
Order Rate Constant ◽  
Competitive Inhibitor ◽  
Binding Studies ◽  
Electrophorus Electricus ◽  
Equilibrium Binding ◽  
Low Ionic Strength ◽  
Hydrolysis Of

The interactions of edrophonium chloride, gallamine triiodide, and propidium diiodide with affinity-purified acetylcholinesterase from Electrophorus electricus have been examined under conditions of low ionic strength (0.001 M Tris, pH 8.0) using kinetic and fluorescence titration techniques. Edrophonium is a competitive inhibitor of the steady-state hydrolysis of acetylthiocholine, with an inhibition constant, Kcomp, of 1.2 × 10−8 M. Double reciprocal plots in the presence of either gallamine or propidium are nonlinear. Similarly, the pre-steady-state carbamoylation of the enzyme by 7-(dimethylcarbamoyloxy)-N-methyl quinolinium iodide is competitively inhibited by edrophonium, whereas the intercepts of the double reciprocal plots of pseudo-first-order rate constant of carbamoylation versus substrate concentration are displaced downwards in the presence of gallamine or propidium. These results, and those of equilibrium binding studies utilizing the fluorescence properties of bound propidium, suggest that gallamine and propidium compete for a peripheral class of anionic sites on the enzyme, whereas edrophonium binds to the anionic subsite of the catalytic site. The characteristics of propidium binding to the eel enzyme differ from those previously observed with enzyme isolated from Torpedo californica. Whereas the tetrameric Torpedo enzyme possesses four binding sites of equal affinity for propidium, the eel enzyme appears to have two classes of propidium binding site. One set of approximately two sites per tetramer is characterized by a dissociation constant of approximately 2–5 × 10−8 M; a second set of two sites bind propidium with a dissociation constant of 4 × 10−6 M. Possible reasons for these differences are discussed.

Sign in / Sign up

Export Citation Format

Share Document