Chymotrypsin inhibitor from potatoes: interaction with target enzymes

Eddy, Jerry Lee; Derr, Judith E.; Hass, George M.

doi:10.1016/0031-9422(80)85105-3

Cited by 6 publications

(5 citation statements)

References 25 publications

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“…Although reactive site cleavage (Laskowski & Sealock, 1971) has not been effected for either PCI-I (Eddy et al, 1980) or PTI, the Arg-38-Asn-39 peptide bond has been identified as the reactive site of the trypsin inhibitor from eggplant (Richardson, 1979). This assignment is consistent with our identification of Leu-38 as the corresponding P] position in PCI-I, a chymotrypsin inhibitor, and of Arg-38 as the P, position in PTI, a trypsin inhibitor.…”

Section: Discussionsupporting

confidence: 86%

Primary structures of two low molecular weight proteinase inhibitors from potatoes

Gm¹,

Ma²,

Ca³

et al. 1982

Biochemistry

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The amino acid sequences of two low molecular weight proteinase inhibitors from Russet Burbank potatoes have been determined. One of these, a chymotrypsin inhibitor, is a peptide of 52 amino acid residues, while the second inhibitor, which is specific for trypsin, contains 51 amino acid residues. These peptides are highly homologous, differing at only nine positions. At position 38, the chymotrypsin inhibitor possesses leucine and the trypsin inhibitor an arginine. This difference probably represents the P1 sites, which are consistent with the respective specificities of the two inhibitors. The inhibitors are also homologous with potato inhibitor II and with an inhibitor previously isolated from eggplants.

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Section: Discussionsupporting

confidence: 86%

Primary structures of two low molecular weight proteinase inhibitors from potatoes

Gm¹,

Ma²,

Ca³

et al. 1982

Biochemistry

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“…We did not attempt to compare this result with that of PI-2 purified from potato, because potato PI-2 is a mixture of several protomers, all with slightly different specificities, which are refractory to separation [5]. Published values for some isolates of PI-2 suggest a K i for chymotrypsin of 20 nm [5] or 0.16 nm [35], but these values probably both account for a mixture of PI-2 protomers with unknown sequence.…”

Section: Variantmentioning

confidence: 98%

Characterization of potato proteinase inhibitor II reactive site mutants

Beekwilder

Schipper

Bakker

et al. 2000

European Journal of Biochemistry

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Potato proteinase inhibitor II (PI-2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI-2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (K i 0.4 nm) and chymotrypsin (K i 0.9 nm), and is quite robust towards mutations at positions P2 or P1H . In contrast, the first reactive site inhibits only chymotrypsin (K i 2 nm), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.Keywords: mutational analysis; Pichia pastoris; serine proteinase inhibitor; Solanum tuberosum.Protease inhibitors are abundant proteins in the storage organs and seeds of plants [1]. In addition, their synthesis is induced to high levels in response to stress, infection and wounding [2]. Potato expresses many different proteinase inhibitors belonging to a wide range of inhibitor families. Members of the potato proteinase inhibitor II (PI-2) family have been shown to inhibit serine proteases, such as trypsin, chymotrypsin, subtilisin, oryzin and elastase [3,4]. Until now, they have been found only among the Solanaceae. Proteins and messenger RNAs have been identified in potato tubers [5,6], wounded tomato and tobacco leaves [4,7], eggplant fruits [8], green tomatoes [9] and ornamental tobacco flower stigma [10]. There is medical interest in the properties of PI-2. It has been shown to have anticarcinogenic properties, protecting mouse embryo fibroblasts from radiation-induced transformation [11]. Although the mechanism underlying this effect is poorly understood, it is correlated to the capacity of PI-2 to inhibit chymotrypsin-like enzymes [12].Plant protease inhibitors such as PI-2 have been proposed to function as part of the plant defense system [13]. The plant defense role is deduced from observations that proteinase inhibitors in leaves are synthesized in response to wounding [2,14,15] or viral infection [2,16]. In addition, direct evidence for a protective role has been obtained; it was shown that transgenic tobacco plants over-expressing potato PI-2 gained resistance against the tobacco hornworm Manduca sexta [17]. Also, when the PI-2 gene was introduced into rice under its own wound-inducible promoter, plants were protected from the major lepidopteran rice pest, Sesamia inferens [18]. PI-2 probably acts by inhibiting digestive proteases from the insects, thereby restricting the availability of amino acids [13].The anti-nutrient activity of PI-2 has been shown to be overcome by pest insects like Spodoptera exigua [19]. When these larvae are reared on tobacco leaves expressing potato PI-2, the spe...

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“…There have been limited kinetic studies on these inhibitors [154] although reversibility of inhibition was observed, as required for a canonical inhibitor. Other studies have restricted analyses to determination of the equilibrium constant (Ki), which in the case of dimeric inhibitors represents a composite of two independent and differently interacting sites.…”

Section: Ppi-2 Family (I20 [4])mentioning

confidence: 96%

“…The P 1 arginine of several inhibitors has been altered to alanine or valine to create elastase inhibitors [73,89,95,113] and to phenylalanine to make chymotrypsin inhibitors [73,89]. [169] RTI-III I18 Brassica napus Trypsin 3.0 x 10 -9 [168] PI II I20 Solanum tuberosum Chymotrypsin 6.0 x 10 5 9.2 x 10 -5 [154] PI II I20 Lycopersicon esculentum Chymotrypsin 8 x 10 -8 [155] While numerous studies have measured the equilibrium dissociation constant for squash inhibitor-proteinase interactions, only a single study has determined kinetic constants [114] (see Table 2). …”

Section: Squash Family (I7 [4])mentioning

confidence: 99%

“…There is considerable reduction in flexibility of the loop on binding [153] and no inhibitor cleavage is observed [145,154]. Reactive site variation is observed across the P 2 -P 1 ' residues with the two outer contact residues being half cystine residues involved not only in loop stabilisation but in holding the two chain variants together [146].…”

Section: Ppi-2 Family (I20 [4])mentioning

confidence: 99%

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