Penicillopepsin: 2.8 a Structure, Active Site Conformation and Mechanistic Implications

Hsu, I-Nan; Delbaere, L. T. J.; James, Michael N.G.; Hofmann, Theo

doi:10.1007/978-1-4757-0719-9_5

Cited by 18 publications

(10 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The movement of the flap eliminates the possible role of proton donor for the p-hydroxyl group of Tyr-75 (7,14,15,26). An earlier study (7) that involved binding 1,2-epoxy-3-p-nitrophenoxypropane to penicillopepsin elicited a movement of the side chain of Tyr-75 towards the active site, suggesting that Tyr-75 played a role of proton donor to the leaving group nitrogen in the catalytic mechanism ofaspartyl proteinases (15,26).…”

Section: Resultsmentioning

confidence: 99%

“…The 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.…”

Section: Methodsmentioning

confidence: 99%

“…The enzyme, penicillopepsin, was prepared, purified, and crystallized as described (6,7,10). Crystals of the native enzyme Least-squares refinement of the initial model deduced at 2.8-A resolution (6, 7) was done with the restrained parameter refinement program of Hendrickson and Konnert (11).…”

Section: Methodsmentioning

confidence: 99%

“…The chemical formula of pepstatin is: 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.…”

mentioning

confidence: 99%

See 3 more Smart Citations

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.

158

123

View full text Add to dashboard Cite

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.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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.

158

123

View full text Add to dashboard Cite

show abstract

“…In mammalian cathepsin D, the Y75 flap is flexible and partially covers the active site (Guha and Padh, 2008;Metcalf and Fusek, 1993). The phenolic-OH group of Tyr 75 donates proton to the amide nitrogen of the scissile bond of the substrate (Hsu et al, 1977). The highly conserved asparagine residue (Asn67 porcine pepsin numbering) found in crustacean cathepsin D (Rojo et al, 2010a), points toward the presence of a N-glycosylation tag for lysosome import as described for other cathepsin D enzymes.…”

Section: Discussionmentioning

confidence: 99%

Is digestive cathepsin D the rule in decapod crustaceans?

Martinez-Alarcon

Saborowski

Rojo‐Arreola

et al. 2018

Comparative Biochemistry and Physiology Part B: Biochemistry an

View full text Add to dashboard Cite

A B S T R A C TCathepsin D is an aspartic endopetidase with typical characteristics of lysosomal enzymes. Cathepsin D activity has been reported in the gastric fluid of clawed lobsters where it acts as an extracellular digestive enzyme. Here we investigate whether cathepsin D is unique in clawed lobsters or, instead, common in decapod crustaceans. Eleven species of decapods belonging to six infraorders were tested for cathepsin D activity in the midgut gland, the muscle tissue, the gills, and when technically possible, in the gastric fluid. Cathepsin D activity was present in the midgut gland of all 11 species and in the gastric fluid from the seven species from which samples could be taken. All sampled species showed higher activities in the midgut glands than in non-digestive organs and the activity was highest in the clawed lobster. Cathepsin D mRNA was obtained from tissue samples of midgut gland, muscle, and gills. Analyses of deduced amino acid sequence confirmed molecular features of lysosomal cathepsin D and revealed high similarity between the enzymes from Astacidea and Caridea on one side, and the enzymes from Penaeoidea, Anomura, and Brachyura on the other side. Our results support the presence of cathepsin D activity in the midgut glands and in the gastric fluids of several decapod species suggesting an extracellular function of this lysosomal enzyme. We discuss whether cathepsin D may derive from the lysosomal-like vacuoles of the midgut gland B-cells and is released into the gastric lumen upon secretion by these cells.

show abstract

Evolution in the structure and function of aspartic proteases

Tang

Wong

1987

J of Cellular Biochemistry

291

194

View full text Add to dashboard Cite

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.

show abstract

Penicillopepsin: 2.8 a Structure, Active Site Conformation and Mechanistic Implications

Cited by 18 publications

References 35 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.

Is digestive cathepsin D the rule in decapod crustaceans?

Evolution in the structure and function of aspartic proteases

Contact Info

Product

Resources

About