Amino acid sequence of a carboxypeptidase inhibitor from tomato fruit

Gm, Hass; Ma, Hermodson

doi:10.1021/bi00511a029

Cited by 47 publications

(38 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This binding stage would immediately precede entry of the carboxy-terminal residue into the S'1 pocket. Because glycine-39 is unnecessary for PCI binding to CPase A (17) it is possible that the CPase A-PCI structure actually represents a complex that is trapped in this intermediate binding stage and is unable to proceed to the true catalytic transition state due to steric interactions between CPase A and the remainder ofthe PCI molecule.…”

Section: Resultsmentioning

confidence: 99%

Binding of ligands to the active site of carboxypeptidase A.

Rees¹,

Lipscomb²

1981

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

View full text Add to dashboard Cite

We compare the detailed binding modes of the 39-amino acid inhibitor from potatoes, glycyl-L-tyrosine, the ester analogue CH30C6H4(CO)CH2CH(CO0)C6H5, and indole acetate to the exopeptidase carboxypeptidase A (EC 3.4.17.1). In the potato inhibitor, cleavage of the COOH-terminal glycine-39 leaves a new carboxylate anion ofvaline-38 having one oxygen on zinc and the other as a receptor of a hydrogen bond from tyrosine-248 of carboxypeptidase. Tyrosine-248 also receives a hydrogen bond from the amide proton of the originally penultimate peptide bond between tyrosine-37 and valine-38. This hydrogen bond suggests product stabilization which is available to peptides and depsipeptides but not to esters lacking an equivalent peptide bond (nonspecific esters). Also, this structure may represent the intermediate binding step for the uncleaved substrate as it moves along the binding subsites. In particular, this may be the binding mode for the substrate after association of the COOH-terminal region of the substrate with the residues at binding subsite S2 (tyrosine-198, phenylalanine-279, and arginine-71) and preceding entry into the catalytic site S;. These stabilized complexes allow some understanding of the effect of indole acetate, shown here to bind in the pocket at S' , as a competitive inhibitor for esters (for which entry into S' precedes the rate-determining catalytic step for hydrolysis) and as a noncompetitive inhibitor for peptides (for which entry into S' is rate limiting). These results, including the binding mode of the ester analogue, are consistent with the original proposal from x-ray studies that both esters and peptides are cleaved with the carboxy terminus at S';, although not necessarily by the same chemical steps.

show abstract

Section: Resultsmentioning

confidence: 99%

Binding of ligands to the active site of carboxypeptidase A.

Rees¹,

Lipscomb²

1981

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

View full text Add to dashboard Cite

show abstract

“…Nevertheless, the results obtained in this work suggest that in case of SmCI N23A, a nonglycosylated form of the protein by elimination of the sugar moiety, it displays an increment of its trypsin inhibitory capability, probably reflecting a better enzyme-inhibitor fit. Aligned inhibitors, SmCI (PCPI_SABMA), carboxypeptidase inhibitor from S. magnifica; TCI (TCI1_RHIBU), carboxypeptidase inhibitor from R. bursa (9); HITCI (A8C364_HAELO), carboxypeptidase inhibitor from H. longicornis (10); MPCI (MCPI_SOLLC) carboxypeptidase inhibitor from Solanum lycopersicum (6); PCI (MCPI_SOLTU) carboxypeptidase inhibitor from Solanun tuberosum (3,4); LCI (MCPI_HIRME) carboxypeptidase inhibitor from H. medicinalis (8); Ascaris carboxypeptidase inhibitor (ACI) (ICAA_ASCSU), carboxypeptidase inhibitor from A. suum (7). Similar or identical residues among the C-terminal amino acid residues of the inhibitors are shaded.…”

Section: Discussionmentioning

confidence: 99%

“…K i values of the inhibitors against different enzymes were determined using the same kinetic strategy and substrates employed for the natural inhibitor. For the pancreatic elastase inhibitory assay, N-Suc-(Ala) 3 -pNA was used as substrate in case of rSmCI (41).…”

Section: Methodsmentioning

confidence: 99%

“…The biological actions of many proteases are controlled by their interaction with specific proteinaceous inhibitors. Unlike endoproteases, for which numerous examples of protein inhibitors have been reported, naturally occurring metallocarboxypeptidase inhibitors are somewhat limited, and so far, they have only been identified in Solanacea, tomato, and potato (PCI) (3)(4)(5), the intestinal parasite Ascaris suum (ACI, Ascaris carboxypeptidase inhibitor) (6,7), the medicinal leech Hirudo medicinalis (LCI) (8), the ticks Rhipicephalus bursa (TCI) (9) and Hemaphysalis longicornis (H1TCI) (10), and in rat and human tissues (latexin or endogenous carboxypeptidase inhibitor (ECI)) (11,12).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica

Rivero

Trejo

Reytor

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

“…The biological action of MCPs is specifically modulated through protein inhibitors. To date, seven such MCP inhibitors have been described from potato and tomato (PCI and MCPI; 38 and 39 residues, respectively) (21,22), medical leech Hirudo medicinalis (LCI; 66 residues) (23), the ticks Rhipicephalus bursa and Haemaphysalis longicornis (TCI and HlTCI; 75 and 77 residues, respectively) (24,25), rat and human latexin (alias ECI; 222 and 223 residues, respectively) (26,27), and the intestinal parasites A. lumbricoides and A. suum (ACI) (12,13). Although the former inhibitors have been studied extensively in terms of activity and structure, ACI has hitherto only been studied for its amino acid sequence.…”

mentioning

confidence: 99%

Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite

Sanglas

Aviles

Huber

et al. 2009

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

View full text Add to dashboard Cite

Roundworms of the genus Ascaris are common parasites of the human gastrointestinal tract. A battery of selective inhibitors protects them from host enzymes and the immune system. Here, a metallocarboxypeptidase (MCP) inhibitor, ACI, was identified in protein extracts from Ascaris by intensity-fading MALDI-TOF mass spectrometry. The 67-residue amino acid sequence of ACI showed no significant homology with any known protein. Heterologous overexpression and purification of ACI rendered a functional molecule with nanomolar equilibrium dissociation constants against MCPs, which denoted a preference for digestive and mast cell A/B-type MCPs. Western blotting and immunohistochemistry located ACI in the body wall, intestine, female reproductive tract, and fertilized eggs of Ascaris, in accordance with its target specificity. The crystal structure of the complex of ACI with human carboxypeptidase A1, one of its potential targets in vivo, revealed a protein with a fold consisting of two tandem homologous domains, each containing a ␤-ribbon and two disulfide bonds. These domains are connected by an ␣-helical segment and a fifth disulfide bond. Binding and inhibition are exerted by the C-terminal tail, which enters the funnel-like active-site cavity of the enzyme and approaches the catalytic zinc ion. The findings reported provide a basis for the biological function of ACI, which may be essential for parasitic survival during infection.ascariasis ͉ crystal structure ͉ host resistance ͉ immunolocalization ͉ metallocarboxypeptidase inhibitor M ore than a quarter of the human population is affected by soil-transmitted helminthes, which impair nutrition and the immune response toward widespread pandemics such as AIDS and tuberculosis (1, 2). The roundworm Ascaris lumbricoides is the most common human parasite of the gastrointestinal tract. It causes ascariasis (3), which has a worldwide distribution with highest prevalence in tropical and subtropical regions and in areas with inadequate sanitation. Ascariasis is triggered by the ingestion of parasite eggs. These evolve to larvae that migrate through different tissues and return to the small intestine, where they mature to adult male and female worms. At this stage, females deposit thousands of eggs daily, which are secreted with the feces, thus contributing to soil contamination and spreading of the infection [for details, see supporting information (SI) Fig. S1]. During its life cycle, Ascaris threatens human health with nonspecific abdominal symptoms, intestinal obstruction and perforation, biliary colic, gallstone formation, liver abscesses, pancreatitis, and pulmonary eosinophilia (4, 5). A nearly identical nematode species, Ascaris suum, is found in the pig. It has a tremendous impact on livestock farming and can also infect primates and humans, giving rise to a similar disease pattern to A. lumbricoides (6-8).As part of the parasite defense strategy, Ascaris roundworms secrete a series of inhibitors to target digestive and immunerelated host proteases, among others pepsi...

show abstract

Amino acid sequence of a carboxypeptidase inhibitor from tomato fruit

Cited by 47 publications

References 22 publications

Binding of ligands to the active site of carboxypeptidase A.

Binding of ligands to the active site of carboxypeptidase A.

Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica

Mammalian metallopeptidase inhibition at the defense barrier of Ascaris parasite

Contact Info

Product

Resources

About