Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products

Wang, T T; Hofmann, Theo

doi:10.1042/bj1530691

Cited by 30 publications

(8 citation statements)

References 22 publications

(24 reference statements)

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“…These residues, Asp-Val-Pro-Thr, could adopt a conformation resembling a flap that could interact preferentially with residues P', P', and P3 ofa substrate, thus providing an explanation for the efficiency of the amino-transfer reactions of pepsin. However, the conformational changes observed may not be sufficient to account for the failure of the intermediates in the transpeptidation reactions to exchange their acyl or amino moieties with the corresponding free amino acids in the reaction mixture (30 …”

Section: Resultsmentioning

confidence: 99%

“…The falloff of intensity of the diffraction pattern due to radiation damage was corrected by a function of time and 0 (18). In this way, 30,000 reflections were measured from one crystal ofthe complex during a period of31 days. The maximum decay correction required for reflections with sinG/A : 0.264 was 55%, whereas that for the data of low sinG/A was only 15-20%.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

James¹,

Sielecki²,

Salituro³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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BiochemistryConformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin ( Communicated by Joseph S. Fruton, June 21, 1982 ABSTRACT Crystals of the molecular complex between the esterified tripeptide fragment of pepstatin and the aspartyl proteinase penicillopepsin are isomorphous with crystals of native penicillopepsin. The difference electron-density map at 1.8-A resolution, computed by using the amplitude differences and refined phases of reflections from the crystal of native penicillopepsin, unambiguously showed the binding mode of isovaleryl Pepstatin is a naturally occurring inhibitor of aspartyl proteinases (1); it has the amino acid composition isovaleryl (Iva)-ValVal-Sta-Ala-StaOH, in which Sta is the residue of statine [(4S,3S)-4-amino-3-hydroxyl-6-methylheptanoic acid] (2). The interaction ofpepstatin with aspartyl proteases is characterized by small dissociation constants [i.e., 4.6 x 10-11 M with porcine pepsin (3) and 1.5 x 10-10 M with penicillopepsin (unpublished results)]. It Structure I Penicillopepsin is an aspartyl proteinase isolated from the mold Penicillium janthinellum. Its crystal structure (6, 7) has been refined at 1.8-A resolution (8) to a conventional R-factor § of 0.136 for those reflections with I, the intensity of Bragg reflections, equal to or greater than the estimated standard deviation of the intensity measurement derived from counting statistics [I 2 o-(I)]. The aspartyl proteinases are characterized by a long binding cleft that can accommodate 7 or 8 amino acid residues of an oligopeptide substrate in an extended conformation. The two catalytically important aspartyl residues, Asp&jand Asp-213, are centrally located in this binding cleft (Asp32and Asp-215 in the porcine pepsin sequence numbering).Fluorescence measurements on the aspartyl proteinases with specific substrates have indicated that conformational mobility of groups in the active site may play an important role in the mechanism of these enzymes (9). MATERIALS AND METHODSThe enzyme, penicillopepsin, was prepared, purified, and crystallized as described (6,7,10). Crystals of the native enzyme The pepstatin derivative Iva-Val-Val-StaOEt was synthesized by the following procedure. t-Butoxycarbonyl (Boc)-StaOEt (12) was deprotected with 4 M HCl in dioxane and sequentially coupled with Boc-L-valine, Boc-L-valine, and isovaleryl anhydride by using procedures previously described for-the synthesis of pepstatin analogues (13) 6137The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

James¹,

Sielecki²,

Salituro³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

158

123

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“…Such reactions, well documented for endoproteases (4,13,28), would be of great interest in CPD-Y catalyzed peptide synthesis. In the present paper the results of such studies, employing peptide amides and peptides as substrates, are reported.…”

Section: Introductionmentioning

confidence: 96%

Carboxypeptidase Y catalyzed transpeptidations and enzymatic peptide synthesis

Breddam

Widmer

Johansen

1980

Carlsberg Res. Commun.

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It is shown that carboxypeptidase Y, in addition to its known amidase and peptidase activities, also exhibits peptidyl-amino-acid-amide hydrolase activity, i.e., amino acid amides are released from peptide amides. These three activities were investigated as to their usefulness in catalyzing transpeptidation reactions in the presence of suitable nucleophiles. Using peptides and peptide amides as substrates and amino acids or amino acid amides as nuc[eophiles, it could be shown that transpeptidation products were formed in accordance to each of the above mentioned activities of carboxypeptidase Y. While transpeptidation via the amidase activity results in an elongation of the peptide chain, transpeptidation via peptidase activity results in the exchange of the C-terminal amino acid residue. If the substrate is a peptide amide, transpeptidation via the peptidyl-amino-acid-amide hydrolase activity will result in the formation of a peptide where the C-terminal amino acid amide is replaced by either an amino acid or an amino acid amide.As conditions have not yet been found where a[t three activities are well separated from each other, it is not generally possible to direct these transpeptidations towards a single desired product in high yield. However, the data presented in this paper indicate that for peptides of certain unique amino acid sequences the carboxypeptidase Y catalyzed transpeptidations may be applicable in the field of enzymatic peptide synthesis.Abbreviations: Ac = acetyl: Bz = benzoyl: CBZ = carbobenzoxy: CPD-Y = carboxypeptidase Y; DMF = dimethylformamide; FA = 3-(2-Furylacryloyl)-; HPLC = high pressure liquid chromatography; NaAc = sodium acetate: TEAF = triethylammonium formate; TEAP = triethylammonium phosphate buffer: All other abbreviations of amino acids, amino acid derivatives and peptides are according to the guideline of the IUPAC-IUB Commission on Biochemical Nomenclature.

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“…Phe-Leu amide, Cbz-Leu-Leu amide and Cbz-Leu-Leu and a number of other dipeptide amides did not serve as substrates (Whitaker and Caldwell 1973) indicating that the enzyme probably recognizes a free amino terminal leucine specifically. Tyr-Leu amide is as good a substrate for penicillopepsin as is Leu-Tyr amide (Wang and Hofmann 1977) while pig pepsin does not hydrolyze Tyr-leu amide at all (Wang and Hofmann 1976b) but cleaves Leu-Tyr amide readily.…”

Section: Endothiu Prasitica Protease Shows Hysteretic Behavior Under mentioning

confidence: 97%

“…In the discussion, it will be assumed that E. parasitica protease is a carboxyl protease (that is, its activity is dependent on one or more carboxyl groups in correct state of ionization) similar to pepsin, rennin and penicillopepsin since it is inhibited by diazoacetyl norleucine methyl ester as are those enzymes (Hofmann 1974). Binding of substrate will be discussed in terms of specific subsites for interaction of the individual amino acid residues within the binding locus as proposed first for papain by Berger and Schechter (1970) and shown for pepsin (Sampath-Kumar and Fruton 1974;Wang and Hofmann 1976b) and penicillopepsin . This model is shown diagrammatically in Fig.…”

Section: Endothiu Prasitica Protease Shows Hysteretic Behavior Under mentioning

confidence: 99%

Acyl Transpeptidation and Hydrolytic Reactions Catalyzed by Endothia Parasitica Protease With Small Peptide Substrates

Lee

Whitaker

1979

J Food Biochemistry

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Endothia parasitica protease shows hysteretic behavior (a lag phase) when acting upon L-leucyl-L-leucine amide at pH 4.5. Initially, the products are L-leucyl-L-leucine, L-leucyl-L-leucyl-L-leucine and L-leucine amide with little formation of L-leucine and no ammonia. The relative concentration ofL-leucyl-L-leucine is about one-fourth that ofL-leucine amide. As the reaction continues the relative concentration of L-leucine increases slowly until it is equal to the concentration of L-leucyl-L-leucine near the end of the reaction with each being one-fourth that of L-leucine amide. Formation of these products can only be explained by a combination of transpeptidation and hydrolytic reactions. By itself, L-leucyl-L-leucine is cleaved very slowly but when mixed with L-leucyl-L-leucine amide the rate is several fold greater than the combined rates of the two alone. L-Leucyl-L-leucyl-L-leucine and L-leucyl-L-leucyl-L-leucine amide as substrates of E. parasitica protease give normal kinetic behavior with no evidence of formation of higher molecular weight compounds. Addition of as little as 1 pM L-leucyl-L-leucyl-L-leucine (Km = 0.348 mM) to a reaction containing 1 mM L-leucyl-L-leucine amide increases the rate of reaction with a shortening of the lag phase. Addition of 100 pM L-leucyl-L-leucyl-L-leucine to such a reaction eliminates the hysteretic behavior. It is proposed that E. parasitica protease is initially in an inactive form which can be activated by substrates as small as L-leucyl-L-leucyl-L-leucine but not by L-leucyl-Lleucine amide. During the lag phase observed with substrates such as L-leucyl-L-leucine amide, the enzyme produces, via my1 transpptidation, larger compounds which can rapidly activate the enzyme. While in the activated form, E. parasitica protease cleaves L-leucyl-L-leucine amide via normal kinetic behavior; however, both acyl transpptidution and hy-53 54 SHIH-LO LEE AND JOHN R. WHITAKER drolysis still occur as shown by the relative concentrations of L-leucine, L-leucyl-L-leucine and L-leucine amide of 1:1:4 near end of the reaction. E. parasitica protease cannot cleave carbobenzoxy-L-leucyl-L-leucine indicating that, at least with this substrate, the enzyme cannot perform amino transpeptidation reactions.CD spectra were measured on a Cary 60 spectrometer using a cuvette with light path of 1 cm and a full range of ellipticity of 0.02 degree. The signal output was fed to a PDPSE computer which averaged the signal and printed out the readings at intervals of one nm (with 10% error). The CD spectrum of the buffer (0.1 M sodium acetate buffer, pH 4.51, taken first, was subtracted from the spectrum of the enzyme in solution automatically by the computer. The spectrum of E. parasitica protease was taken at a final concentration of 20 pM in a volume of 3 ml in a cylindrical cuvette in the presence and absence of Leu-Leu amide.

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Acyl and amino intermediates in reactions catalysed by pig pepsin. Analysis of transpeptidation products

Cited by 30 publications

References 22 publications

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

Conformational flexibility in the active sites of aspartyl proteinases revealed by a pepstatin fragment binding to penicillopepsin.

Carboxypeptidase Y catalyzed transpeptidations and enzymatic peptide synthesis

Acyl Transpeptidation and Hydrolytic Reactions Catalyzed by Endothia Parasitica Protease With Small Peptide Substrates

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