The Inactivation of Penicillopepsin with l,2-Epoxy-3-(p-nitrophenoxy)propane, an Active-Site Directed Reagent

1974 ◽  
Vol 52 (11) ◽  
pp. 1018-1023 ◽  
Author(s):  
G. Mains ◽  
T. Hofmann
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Side Chain ◽  
Porcine Pepsin ◽  
Protein Inactivation ◽  
Pepsin Inhibitor

Penicillopepsin was fully inactivated by the pepsin inhibitor 1,2-epoxy-3-(p-nitrophenoxy) propane, and 1.3 ± 0.3 mol of reagent became associated with each mole of protein. Inactivation was more rapid at pH 3.0 than at pH 6.0. Approximately 1 equivalent of the bound reagent was esterified to an aspartic acid side chain. Enzyme previously inactivated with diazoacetylnorleucine methyl ester did not react with the epoxide; and enzyme that was first inactivated with the epoxide did not react with the diazo inhibitor. The results add further evidence for the enzymatic similarity of porcine pepsin and penicillopepsin.

scholarly journals An active-site peptide from pepsin C

1971 ◽  
Vol 123 (1) ◽  
pp. 75-82 ◽  
Author(s):  
J. Kay ◽  
A. P. Ryle
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Carboxyl Group ◽  
Active Site Residue ◽  
Aspartic Acid Residue ◽  
Porcine Pepsin ◽  
Ph Values ◽  
Complete Inactivation ◽  
Active Site Sequence

Porcine pepsin C is inactivated rapidly and irreversibly by diazoacetyl-dl-norleucine methyl ester in the presence of cupric ions at pH values above 4.5. The inactivation is specific in that complete inactivation accompanies the incorporation of 1mol of inhibitor residue/mol of enzyme and evidence has been obtained to suggest that the reaction occurs with an active site residue. The site of reaction is the β-carboxyl group of an aspartic acid residue in the sequence Ile-Val-Asp-Thr. This sequence is identical with the active-site sequence in pepsin and the significance of this in terms of the different activities of the two enzymes is discussed.

Amino acid sequence around the active site aspartic acid in penicillopepsin

1970 ◽  
Vol 48 (9) ◽  
pp. 1014-1016 ◽  
Author(s):  
J. Šodek ◽  
T. Hofmann
Keyword(s):  
Amino Acid ◽  
Methyl Ester ◽  
Amino Acid Sequence ◽  
Aspartic Acid ◽  
Strong Evidence ◽  
Active Site ◽  
Ester Group ◽  
Acid Proteinase ◽  
Penicillium Janthinellum ◽  
Porcine Pepsin

Penicillopepsin, the acid proteinase of Penicillium janthinellum, was specifically inactivated with diazoacetylnorleucine methyl ester. The peptide containing the glycollylnorleucine methyl ester group was isolated from a peptic digest. The amino acid sequence was found to be Ile∙Ala∙β(glycollyl-Nle OMe)-Asp∙Thr∙Gly∙Thr∙Thr∙Leu and is thus almost identical with the active site peptide of porcine pepsin: Ile∙Val∙Asp∙Thr∙Gly∙Thr∙Ser. This finding provides strong evidence for an evolutionary homology between penicillopepsin and porcine pepsin.

scholarly journals An aspartic acid residue at the active site of pepsin. The isolation and sequence of the heptapeptide

1969 ◽  
Vol 113 (2) ◽  
pp. 377-386 ◽  
Author(s):  
R. S. Bayliss ◽  
J. R. Knowles ◽  
Grith B. Wybrandt
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Irreversible Inhibitor ◽  
Aspartic Acid Residue ◽  
Phenylalanine Methyl Ester

Pepsin reacts stoicheiometrically with the active-site-directed irreversible inhibitor N-diazoacetyl-l-phenylalanine methyl ester, with concomitant loss of all proteolytic and peptidolytic activity. The reagent esterifies a unique aspartic acid residue in pepsin, which is in the sequence:Ile-Val-Asp-Thr-Gly-Thr-Ser

scholarly journals Bovine pepsinogens and pepsins. The sequence around a reactive aspartyl residue

1971 ◽  
Vol 124 (4) ◽  
pp. 673-676 ◽  
Author(s):  
Patricia A. Meitner
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Specific Inhibitor ◽  
Enzymic Activity ◽  
Active Centre ◽  
Aspartic Acid Residue ◽  
Porcine Pepsin ◽  
Simultaneous Loss ◽  
Aspartyl Residue

The specific inhibitor, N-diazoacetylnorleucine methyl ester reacts stoicheiometrically with bovine pepsin resulting in a simultaneous loss of all enzymic activity. A peptide containing a modified aspartyl group was isolated from bovine pepsin labelled with 14C-labelled inhibitor. The aspartic acid residue is presumed to be part of the active centre and is in the same heptapeptide sequence as in porcine pepsin: Ile-Val-Asp-Thr-Gly-Thr-Ser.

Isolation and characterization of a peptide derived from the active site of chicken pepsin

1980 ◽  
Vol 45 (7) ◽  
pp. 2131-2134 ◽  
Author(s):  
Helena Keilová ◽  
Vladimír Kostka ◽  
Miroslav Baudyš
Keyword(s):  
Amino Acid ◽  
Methyl Ester ◽  
Amino Acid Composition ◽  
Aspartic Acid ◽  
Acid Composition ◽  
Active Site ◽  
Its Sequence ◽  
Aspartic Acid Residue ◽  
Isolation And Characterization

A peptide was isolated from chicken pepsin which contains the aspartic acid residue reacting with diazoacetyl-D,L-norleucine methyl ester in the presence of Cu2+ -ions. The peptide is N-terminated with isoleucine and contains (besides isoleucine) valine, aspartic acid, two threonines, serine, and leucine. In concurrent experiments a peptide of the same composition was isolated from the thermolysin digest of chicken pepsin and its sequence determined as Ile-Val-Asp-Thr-Gly-Thr-Ser-Leu. Since both peptides have entirely identical amino acid composition and other characteristics, the sequenced peptide corresponds to the peptide isolated from the active site of the enzyme.

Synthesis of oxytocin analogues modified in the tripeptide side-chain by condensation of aminoterminal linear hexapeptide with the carboxyterminal tripeptide

1979 ◽  
Vol 44 (1) ◽  
pp. 275-287 ◽  
Author(s):  
Jan Hlaváček ◽  
Tomislav Barth ◽  
Karel Bláha ◽  
Karel Jošt
Keyword(s):  
Methyl Ester ◽  
Side Chain ◽  
Glycine Methyl Ester

For the synthesis of oxytocin (Ia) analogues modified in the carboxyterminal part of the molecule, a method based on the condensation of protected aminoterminal hexapeptide with tripeptides by the action of dicyclohexylcarbodiimide and pentafluorophenol in the presence of 1-hydroxybenzotriazole was devised. Using this method [7-[U-13C]proline]oxytocin (Ib), des-9-glycine-oxytocin (Ic) and methyl ester of oxytocinoic acid ([9-glycine methyl ester]oxytocin) (Id) were prepared.

scholarly journals Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

1991 ◽  
Vol 280 (3) ◽  
pp. 659-662 ◽  
Author(s):  
J Martín ◽  
A Slade ◽  
A Aitken ◽  
R Arche ◽  
R Virden
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Iodoacetic Acid ◽  
Penicillin Acylase ◽  
Protein Product ◽  
Side Chain ◽  
Serine Residue ◽  
Conserved Sequence ◽  
Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

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