THE HYDROLYSIS OF PHENYLALANINE METHYL ESTER BY BOVINE THROMBIN

Thrombin, usually regarded as a trypsin-like enzyme capable of hydrolyzing only esters of the amino acids arginine and lysine, was found to hydrolyze certain other amino acid esters which are considered specific chymotrypsin substrates. L-Phenylalanine methyl ester inhibited the activation of purified bovine prothrombin by autoprothrombin C, Ac-globulin, phospholipid, and calcium. It was subsequently shown that thrombin is capable of hydrolyzing L-phenylalanine methyl ester or L-tyrosine ethyl ester. This activity developed simultaneously with fibrogen clotting activity during activation of purified bovine prothrombin in 25% sodium citrate solutions. Moreover, the activity was closely associated with clotting activity on subsequent chromatography on DEAE-cellulose and Amberlite IRC-50 resin columns. All preparations of bovine thrombin, representing a wide range of purity, which have been examined, exhibited this hydrolyzing activity toward L-phenylalanine methyl ester. Further evidence linking this esterase activity with fibrinogen clotting activity was obtained when both activities were inhibited by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, an inhibitor of chymotrypsin.

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Hydrolysis of L-Histidine Methyl Ester

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651211 ◽

1968 ◽

Vol 19 (03/04) ◽

pp. 321-333

Author(s):

E. R Cole

Keyword(s):

Methyl Ester ◽

Esterase Activity ◽

Sodium Citrate ◽

Deae Cellulose ◽

Sodium Cholate ◽

Michaelis Constant ◽

Bovine Thrombin ◽

Optimum Ph ◽

Autoprothrombin C ◽

Hydrolysis Of

SummaryThe hydrolysis of L-histidine methyl ester (HME) by bovine thrombin preparations has been investigated. Activation of purified bovine prothrombin in 25% sodium citrate solution resulted in the simultaneous development of fibrinogen clotting activity and of TAME and HME esterase activities. Most of the HME esterase activity was identified with fibrinogen clotting activity on sequential chromatography of activated prothrombin on DEAE-cellulose and Amberlite CG-50 resin columns, although some HME esterase activity could be demonstrated in concentrates of the autoprothrombin C fraction. The optimum pH for HME hydrolysis by thrombin was found at 7.6 in phosphate and Tris buffered reactions. Tris buffer and other amines depress HME esterase activity of thrombin, while sodium cholate accelerates the reaction. The Michaelis constant, Km, was estimated to be 0.134 M at pH 7.6 in phosphate buffer and at 37° C.

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Comparative studies of the specificities of α-chymotrypsin and subtilisin BPN′. Studies with flexible substrates

Biochemical Journal ◽

10.1042/bj1260645 ◽

1972 ◽

Vol 126 (3) ◽

pp. 645-657 ◽

Cited By ~ 23

Author(s):

T. N. Pattabiraman ◽

W. B. Lawson

Keyword(s):

Methyl Ester ◽

Binding Site ◽

Comparative Studies ◽

Detrimental Effect ◽

Flexible Substrates ◽

Enhancing Effect ◽

Wide Range ◽

Phenylalanine Methyl Ester ◽

Hydrolysis Of ◽

Tryptophan Methyl Ester

A series of arylalkanoate esters and α-acetamidoarylalkanoate esters were tested as substrates for α-chymotrypsin and subtilisin BPN′. Chymotrypsin hydrolysed N-acetyl-l-phenylalanine methyl ester and methyl 4-phenylbutyrate faster than their respective higher and lower homologues, whereas methyl 2-acetamido-6-phenylhexanoate and methyl 6-phenylhexanoate were better substrates for subtilisin than their lower homologues. N-Acetyl-l-tryptophan methyl ester and its analogue, N-acetyl-3-(1-naphthyl)-alanine methyl ester, were hydrolysed 23 times faster by chymotrypsin than by subtilisin. These results indicate that the binding site of α-chymotrypsin is roughly 1.1nm (11Å) long and curved, whereas that of subtilisin is a longer system and less curved. The stereo-specificity during the hydrolysis of typical substrates by both enzymes was found to vary over a wide range. The enhancing effect of the α-acetamido group in the l-series of substrates and the detrimental effect in the d-series of substrates also varies considerably.

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The stereospecificity of subtilisin BPN′ towards 1-keto-3-carbomethoxy-1,2,3,4-tetrahydroisoquinoline

Canadian Journal of Biochemistry ◽

10.1139/o69-155 ◽

1969 ◽

Vol 47 (10) ◽

pp. 985-987 ◽

Cited By ~ 5

Author(s):

Hermann Dugas

Keyword(s):

Amino Acid ◽

Methyl Ester ◽

Active Site ◽

Kinetic Constants ◽

Neutral Amino Acid ◽

Amino Acid Esters ◽

Open Chain ◽

Hydrolysis Of ◽

Acid Esters ◽

Chain Ester

Like α-chymotrypsin, subtilisin BPN′ reverses its normal stereospecificity when 1-keto-3-carbomethoxy-1,2,3,4-tetrahydroisoquinoline is the substrate; the D-isomer is hydrolyzed readily, the L-isomer is not. However, the enzyme retains its normal stereospecificity when the related open-chain ester N-benzoyl-alanine methyl ester is the substrate; the kinetic constants for hydrolysis of the L-isomer are comparable to those for hydrolysis of other neutral amino acid esters. These results suggest that the stereochemical requirements at the active site of subtilisin BPN′ are similar to those of α-chymotrypsin.

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Destruction of intracellular and isolated Leishmania mexicana amazonensis amastigotes by amino acid amides

Parasitology ◽

10.1017/s0031182000058297 ◽

1988 ◽

Vol 96 (2) ◽

pp. 289-296 ◽

Cited By ~ 12

Author(s):

M. Rabinovitch ◽

V. Zilberfarb

Keyword(s):

Amino Acid ◽

Methyl Ester ◽

Methyl Esters ◽

Hydrolytic Enzymes ◽

Amino Acid Esters ◽

Leishmania Mexicana ◽

Acid Methyl ◽

Hydrolysis Of ◽

Acid Esters ◽

Amino Acid Methyl Esters

SummaryL-amino acid esters such as leucine methyl ester (Leu-OMe) destroy Leishmania mexicana amazonensis amastigotes by a mechanism which may involve hydrolysis of the compounds by parasite enzymes. Moreover, several esters (e.g. Ile-OMe) prevent the killing of parasites by Leu-OMe, perhaps by inhibition of the hydrolytic enzymes. We show here that certain amino acid amides are also leishmanicidal. Killing of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by reduction of the tetrazolium MTT. Amino acid amides were generally less active than the methyl esters and several were more toxic to the macrophages, as determined by inspection of Giemsa-stained preparations. Ranks of activity of the amides on isolated amastigotes were Trp > Leu > Phe > Met > Tyr. The amides of Ala, Gly, Val, Ile, His and D-Leu were inactive. This pattern of activity is similar to that of amino acid methyl esters. Ile-NH2 and a few other amides protected intracellular as well as isolated parasites from killing by Leu-OMe. Conversely, Ile-OMe reduced the toxicity of Leu-NH2 for isolated amastigotes. None of the esters or amides assayed prevented the destruction of Leishmania by Trp-NH2. The results are compatible with the view that amino acid esters and amides may be recognized by the same or similar parasite enzymes.

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Synthesis and Complexation Studies of Optically Active Aza- and Diazacrown Ethers Containing a Pyrene Fluorophore Unit

Periodica Polytechnica Chemical Engineering ◽

10.3311/ppch.14467 ◽

2019 ◽

Vol 64 (1) ◽

pp. 20-36

Author(s):

Dávid Pál ◽

Martin Gede ◽

Ildikó Móczár ◽

Péter Baranyai ◽

Péter Bagi ◽

...

Keyword(s):

Methyl Ester ◽

Photoinduced Electron Transfer ◽

Fluorescence Enhancement ◽

Optically Active ◽

Primary Amines ◽

Amino Acid Esters ◽

Diazacrown Ethers ◽

Complexation Studies ◽

Phenylalanine Methyl Ester ◽

Acid Esters

Novel enantiopure azacrown [(R,R)-1 and (S,S)-1] and diazacrown [(R,R)-2–(R,R)-4 and (S,S)-2–(S,S)-4] ethers containing a pyrene fluorophore unit and two phenyl groups at their chiral centers were obtained in multistep syntheses. The action of these chemosensors is based on the photoinduced electron transfer (PET) process, thus they show fluorescence enhancement in the presence of protonated primary amines and amino acid esters. Their recognition abilities toward the enantiomers of 1-phenylethylamine hydrogen perchlorate (PEA), 1-(1-naphthyl) ethylamine hydrogen perchlorate (NEA), phenylglycine methyl ester hydrogen perchlorate (PGME), and phenylalanine methyl ester hydrogen perchlorate (PAME) were examined in acetonitrile using fluorescence spectroscopy.

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