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.

scholarly journals Substrate Inhibition in the Hydrolysis of Hippuric Acid Esters by Carboxypeptidase A

1975 ◽  
Vol 53 (2) ◽  
pp. 283-294 ◽  
Author(s):  
Joe Murphy ◽  
John W. Bunting
Keyword(s):  
Substrate Concentration ◽  
Hippuric Acid ◽  
Substrate Inhibition ◽  
Competitive Inhibitor ◽  
Side Chain ◽  
Carboxypeptidase A ◽  
Substrate Activation ◽  
Hydrolysis Of ◽  
Substrate Concentrations ◽  
Acid Esters

The dependence of initial velocity upon substrate concentration has been examined in the carboxypeptidase A catalyzed hydrolysis of the following hippuric acid esters (at pH 7.5, 25°, ionic strength O.5): C6H5CONHCH2CO2CHRCO2H: R=CH3; CH2CH3;(CH2)2CH3; (CH2)3CH3; (CH2)5CH3; CH(CH3)2; CH2CH(CH3)2; C6H5; CH2C6H5. All of these esters display marked substrate inhibition of their enzymic hydrolyses. With the exception of R=CH3, the velocity-substrate concentration profiles for each of these esters can be rationalized by the formation of an E.S2 complex which, independent of the alcohol moiety of the ester, reacts approximately 25 times more slowly than the E.S complex. For most of these esters, the formation of E.S2 approximates ordered binding of the substrate molecules at the catalytic and inhibitory sites. While binding at the catalytic site is markedly dependent on the nature of the R group, binding of a second substrate molecule to E.S is not significantly affected by the nature of the R side chain. For R=C6H5, the D ester is neither a substrate nor a competitive inhibitor of the hydrolysis of the L-ester but can replace the L-ester at the binding site which is responsible for substrate inhibition. The kinetic analysis suggests that this behavior of D and L -enantiomers is also typical of the other esters examined (except possibly R=CH3). For R=CH3 only, substrate activation also seems to occur prior to the onset of substrate inhibition at higher substrate concentrations.

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.

Reversible Inhibition of Carboxypeptidase A. II. Inhibition of Esterase Activity by Dicarboxylic Acid Anions

1974 ◽  
Vol 52 (11) ◽  
pp. 2053-2063 ◽  
Author(s):  
John W. Bunting ◽  
Chester D. Myers
Keyword(s):  
Dicarboxylic Acid ◽  
Competitive Inhibition ◽  
Dicarboxylic Acids ◽  
Competitive Inhibitor ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Monocarboxylic Acids ◽  
Reversible Inhibition ◽  
Competitive Inhibitors ◽  
Hydrolysis Of

Reversible inhibition of the hydrolysis of O-hippuryl-L-3-phenyllactic acid by carboxypeptidase A has been studied for a series of decarboxylic acids at 25°, pH 7.5, and ionic strength 0.2. All inhibitors studied displayed either strictly competitive or partially competitive inhibition kinetics. For the series CO2H(CH2)nCO2H, strictly competitive inhibition was observed for n = 1, 3, 4, 8, 10, whereas partially competitive inhibition occurs for n = 2, 5, 6, 7. A series of 11 alkyl- and aryl-substituted malonic acids were all strictly competitive inhibitors; for a series of six alkylmalonic acids the inhibition constants are correlated with the Hansch π-parameter by the equation –log K1 = 2.257π + 1.75; arylmalonic acids are poorer inhibitors than expected on the basis of their π-parameters, in accord with a similar observation for monocarboxylic acids. Phthalic acid is a strictly competitive inhibitor (K1 = 1.7 mM), whereas the isomeric isophthalic and terephthalic acids cause relatively little inhibition even at 0.1 M; maleic acid is a partially competitive inhibitor, whereas the isomeric fumaric acid gives only 15% inhibition at 0.1 M. Homophthalic acid and 2,2-dimethyl- and 3,3-dimethylglutaric acids were also investigated.The characteristics of partially competitive inhibition displayed by all dicarboxylic acids and also monomethyl succinate and succinamic acid are consistent with a scheme which assumes the formation of an E.I2 complex. The observed specificity of dicarboxylic acid binding is used to postulate a schematic diagram for binding of these species to the enzyme, and an interpretation of this diagram is suggested on the basis of the crystallographically determined structure of the enzyme.

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.

Effect of ethanol on peptidases of hamster jejunal brush-border membrane

1982 ◽  
Vol 242 (5) ◽  
pp. G442-G447
Author(s):  
P. K. Dinda ◽  
I. T. Beck
Keyword(s):  
Conformational Changes ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Kinetic Studies ◽  
Inhibitory Effect ◽  
Time Dependent ◽  
Peptidase Activity ◽  
Dependent Manner ◽  
Dose Dependent ◽  
Hydrolysis Of

This study was undertaken to investigate the effect of ethanol on the brush-border activity of the small intestine. Brush-border membrane isolated from hamster jejunum was incubated with L-phenylalanylglycine (Phe-Gly), L-leucylglycine (Leu-Gly), or glycyl-L-tyrosine (Gly-Tyr) in the absence and presence of 1-5% (wt/vol) ethanol, and the L-amino acids liberated were determined. Ethanol was found to depress the hydrolysis of all peptides in a dose-dependent manner. The inhibitory effect of ethanol on the peptidases does not appear to be time dependent. The ethanol-induced inhibition of peptidase activity is completely reversible. Kinetic studies indicate that ethanol caused a decrease in the Vmax of the enzymes responsible for the hydrolysis of the Phe-Gly and Gly-Tyr but did not have any effect on their Km. In the hydrolysis of Leu-Gly, two enzymes were involved, and ethanol depressed the Vmax of both without affecting the Km of either. These findings suggest that ethanol produces conformational changes of the peptidases involved in the hydrolysis of these three dipeptides.

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.

Serum Cholinesterase Variants: Examination of Several Differential Inhibitors, Salts, and Buffers Used to Measure Enzyme Activity

1971 ◽  
Vol 17 (3) ◽  
pp. 183-191 ◽  
Author(s):  
Philip J Garry
Keyword(s):  
Enzyme Activity ◽  
Sodium Fluoride ◽  
Phosphate Buffer ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Serum Cholinesterase ◽  
Low Concentrations

Abstract Dibucaine, used as a differential inhibitor with acetyl-, propionyl-, and butyrylthiocholine as substrate, clearly identified the "usual" and "atypical" serum cholinesterases. Succinylcholine was also used successfully as a differential inhibitor with butyrylthiocholine as substrate. Sodium fluoride, used as a differential inhibitor, gave conflicting results, depending on whether Tris or phosphate buffer was used in the assay. Mono- and divalent cations (NaCl, KCl, MgCl2, CaCl2, and BaCl2) activated the "usual" and inhibited the "atypical" enzyme at low concentrations. The "usual" enzyme had the same activity in 0.05 mol of Tris or phosphate buffer per liter, while the heterozygous and "atypical" enzymes showed 12 and 42% inhibition, respectively, when assayed in the phosphate buffer. Kinetic studies showed the phosphate acted as a competitive inhibitor of "atypical" enzyme. Km values, determined for "usual" and "atypical" enzymes, were 0.057 and 0.226 mmol/liter, respectively, with butyrylthiocholine as substrate.

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.

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