Studies in Bile Salt Solutions. XIV. Electronic, Charge and Steric Substrate-Effects on the Esterase Activity of Bile-Salt-Stimulated Human Milk Lipase. Hydrolysis of 4-Substituted Phenyl Propionates

1986 ◽  
Vol 39 (2) ◽  
pp. 259 ◽  
Author(s):  
CJ Oconnor ◽  
ASH Mitha
Keyword(s):  
Human Milk ◽  
Bile Salt ◽  
Active Site ◽  
Esterase Activity ◽  
Electronic Charge ◽  
Electronic Interaction ◽  
Lipase Hydrolysis ◽  
Rate Determining Step ◽  
Esterolytic Activity ◽  
Hydrolysis Of

The rate constants of hydrolysis of a series of 4-substituted phenyl propionates, catalysed by bile-salt-stimulated human milk lipase in the absence and presence of cholate or taurocholate stimulation, have been measured at pH 7.3, 310.5 K. There is little evidence for an alkyl site electronic interaction in the rate-determining step of the esterolytic reaction. However, a negatively charged substrate or an amido-substituent caused an inhibition of unstimulated esterase activity. In the presence of the bile-salt cofactors, esterolytic activity against charged substrates may be stimulated or inhibited, depending on the proximity of the charge to the steroidal side chain and the subsequent substrate-interaction within the surrounding environment of the active site. It has been confirmed that bile-salt- stimulated lipase is not an amidase , but that an amide, of the correct geometry, may occupy the active site and restrict esterase activity.

Studies in Bile Salt Solutions .XIII. Hydrophobic Substrate Effects on the Esterase Activity of Bile-Salt-Stimulated Human-Milk Lipase. Hydrolysis of 4-Nitrophenyl Alkanoates and Alkyl 4-Nitrobenzoates

1986 ◽  
Vol 39 (2) ◽  
pp. 249 ◽  
Author(s):  
CJ Oconnor ◽  
ASH Mitha ◽  
P Walde
Keyword(s):  
Human Milk ◽  
Bile Salt ◽  
Binding Site ◽  
Sodium Taurocholate ◽  
Sodium Cholate ◽  
Lipase Hydrolysis ◽  
Nitrobenzoic Acid ◽  
Hydrocarbon Chains ◽  
Hydrophobic Nature ◽  
Hydrolysis Of

The pseudo-first-order rate constants of hydrolysis of a series of 4-nitrophenyl alkanoates and a series of n-alkyl esters of 4-nitrobenzoic acid and of 4-nitrophenyl hexahydrobenzoate and cyclohexyl 4-nitrobenzoate, catalysed by bile-salt-stimulated human milk lipase in the absence and presence (2 mmol dm-3) of sodium cholate/cholic acid and sodium taurocholate , have been measured at pH 7.3, 310.5 K. It has been shown that the enzyme possesses a specific esterase acyl binding site which almost completely excludes the binding therein of a cyclohexyl group. There is also present a specific alkyl binding site which can fully accommodate a cyclohexyl ring. Both binding sites are hydrophobic in nature, but although the hydrophobic nature of the alkyl binding site is affected by bile-salt stimulation, that of the acyl site is not. Hydrophobicity parameters have been calculated for hydrocarbon chains lying in the acyl and alkyl binding positions of bile-salt-stimulated human milk lipase.

Clotting And Chromogenic Substrate Assays Measure Separate Thrombin Activities

1981 ◽  
Author(s):  
M P Milad ◽  
H I Hassouna
Keyword(s):  
Protease Inhibitor ◽  
Blood Coagulation ◽  
Active Site ◽  
Esterase Activity ◽  
Synthetic Peptides ◽  
Important Implication ◽  
Antithrombin Iii ◽  
Chromogenic Substrate ◽  
Functional Sites ◽  
Esterolytic Activity

Thrombin, the final serine protease responsible for the conversion of fibrinogen to fibrin, is known to have the ability to cleave other peptides in addition to those involved in blood coagulation. Many synthetic substrates were devised to be acted on by thrombin reflecting esterolytic, amidolytic and other cleaving properties. Antithrombin III, the major protease inhibitor of plasma, has been shown to bind the proteolytic properties of thrombin irreversibly. The purpose of this study was to determine whether all enzymatic properties are destroyed simultaneously or whether the inhibitor binds the active site leaving esterolytic functions intact.Purified α-thrombin was examined with fibrinogen, and H-D-Phe-Pip-arg-p-nitroanilide assays to prepare standard working curves relating units thrombin to the rate of fibrin endpoint or to the rate of production of chromophore. Then progressive antithrombin inactivation of seryl residue was examined by increasing concentrations of ATIII incubated with thrombin for five minutes and aliquots simultaneously examined for the ability to clot a standard fibrinogen solution and to cleave the synthetic substrate.The clotting activity decreased steadily with increasing antithrombin, whereas the esterase activity remained constant. The study provides further evidence to the observations of Chang et al (Biochemistry 18: 113, (1979)) that thrombin contains two functional sites, one highly specific for the fibrinopeptide region of fibrinogen and the second site relatively nonspecific, responsible for thrombin’s esterolytic activity. An important implication of this study is that clotting assays and chemical assays that use synthetic peptides reflect separate functional properties of the enzyme thrombin.

scholarly journals Lipoamidase activity in normal and mutagenized pancreatic cholesterol esterase (bile salt-stimulated lipase)

1993 ◽  
Vol 291 (1) ◽  
pp. 65-69 ◽  
Author(s):  
D Y Hui ◽  
K Hayakawa ◽  
J Oizumi
Keyword(s):  
Enzyme Activity ◽  
Gastrointestinal Tract ◽  
Recombinant Protein ◽  
Human Milk ◽  
Bile Salt ◽  
Enzyme Kinetics ◽  
Cholesterol Esterase ◽  
Amide Hydrolysis ◽  
Ester Linkage ◽  
Hydrolysis Of

Purified human milk lipoamidase was digested with endoproteinase Lys-C and the digested peptides were subjected to gasphase microsequence analysis. The sequencing of three isolated peptides of human milk lipoamidase revealed the identity of this protein with human milk bile salt-stimulated lipase (pancreatic cholesterol esterase). The identity of the cholesterol esterase with lipoamidase was confirmed by expressing a recombinant form of rat pancreatic cholesterol esterase and testing for lipoamidase activity of the recombinant protein. The results showed that the recombinant cholesterol esterase displayed both lipolytic and lipoamidase activities and was capable of hydrolysing triacetin and lipoyl-4-aminobenzoate (LPAB). The mechanisms of the esterase and amidase activities of the enzyme were further tested by determining enzyme activity in a mutagenized cholesterol esterase with a His435-->Gln435 substitution. This mutation has been shown previously to abolish enzyme activity against esterase substrates [DiPersio, Fontaine and Hui (1991) J. Biol. Chem. 266, 4033-4036]. We showed that the mutagenized protein was effective in hydrolysing the amidase substrate LPAB and displayed similar enzyme kinetics to those of the native enzyme. These data indicate that the mechanism for the cholesterol esterase hydrolysis of lipoamides is different from that of the hydrolysis of substrates with an ester linkage. The presence of an enzyme in the gastrointestinal tract capable of both ester and amide hydrolysis suggests an important role for this protein in the digestion and absorption processes.

Studies in Bile-Salt Solutions. XXII. The Effect of Reversed Micelles and of Aerosol-OT Aqueous Micelles on the Esterase Activity of Bile-Salt-Stimulated Human-Milk Lipase. Determination of Enzyme-Inhibitor Complex Dissociation Constants

1986 ◽  
Vol 39 (12) ◽  
pp. 2037 ◽  
Author(s):  
CJ Oconnor ◽  
IC Stockley ◽  
P Walde
Keyword(s):  
Human Milk ◽  
Bile Salt ◽  
Esterase Activity ◽  
Dissociation Constants ◽  
Sodium Taurocholate ◽  
Reaction Medium ◽  
Tris Buffer ◽  
Hexadecyltrimethylammonium Bromide ◽  
Aerosol Ot ◽  
Surfactant Complex

The esterase activity of bile-salt-stimulated human milk lipase has been measured against several 4-nitrophenyl alkanoate esters, including 4-nitrophenyl propionate, in reversed micellar solutions of several surfactants. The reaction media used were Aerosol-OT in isooctane, hexadecyltrimethylammonium bromide in chloroform/n-octane (1 : 1 v/v) or in chloroform, Triton X-100 in carbon tetrachloride or isooctane, lecithin in n-octane or diethyl ether/methanol (95 : 5 v/v) or benzene, and Brij 56 in cyclohexane . The reaction conditions varied over a range of cosolubilized water concentrations and initial pH values of added borate, glycine and Tris buffer solutions. In all cases decreased enzymic activity was observed, even if sodium taurocholate was present in the reaction medium. A detailed examination has been made on the effect of changing concentrations of 4-nitrophenyl propionate and of Aerosol-OT on the esterase activity of bile-salt-stimulated human milk lipase at 298 K in aqueous solutions of Tris buffer at pH 7.5. The inhibition induced by Aerosol-OT was identified as being reversible and mixed. The dissociation constants of the enzyme-surfactant complex and of the (enzyme-ester)-surfactant complex have been calculated to be equal to 0.125 and 0.48 mmol 1-1, respectively.

Effect of Temperature and pH on the Esterase Activity of Bile-Salt-Stimulated Human Milk Lipase

1988 ◽  
Vol 3 (3) ◽  
pp. 219-231 ◽  
Author(s):  
Charmian J. O'Connor ◽  
Paul A.G. Butler ◽  
Bridget M. Sutton
Keyword(s):  
Human Milk ◽  
Bile Salt ◽  
Esterase Activity ◽  
Effect Of Temperature

scholarly journals Bile Salt-Stimulated Lipase and Esterase Activity in Human Milk after Collection, Storage, and Heating: Nutritional Implications

1984 ◽  
Vol 18 (4) ◽  
pp. 382-386 ◽  
Author(s):  
Jean M Wardell ◽  
Andrew J Wright ◽  
William G Bardsley ◽  
Stephen W D'souza
Keyword(s):  
Human Milk ◽  
Bile Salt ◽  
Esterase Activity

scholarly journals Reversal of part of the aldehyde dehydrogenase reaction pathway during the hydrolysis of an ester

1979 ◽  
Vol 183 (2) ◽  
pp. 459-462 ◽  
Author(s):  
R J S Duncan
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Active Site ◽  
Esterase Activity ◽  
Reaction Pathway ◽  
Ester Hydrolysis ◽  
Polyacrylamide Gel ◽  
Rabbit Liver ◽  
Dehydrogenase Reaction ◽  
Hydrolysis Of

An aldehyde dehydrogenase from rabbit liver, a homogeneous protein on three distinct polyacrylamide-gel systems, has an associated 4-nitrophenyl esterase activity. At pH 7.0 in the presence of 80 micrometer-NADH and 800 micrometer-4-nitrophenyl acetate the enzyme produces NAD+ and a stoicheiometric amount of an aldehyde, as well as hydrolysing the ester. On this and other evidence it is proposed that ester hydrolysis occurs at the usual active site of the enzyme.

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