Enteropeptidase: Structure, Function, and Application in Biotechnology

Mikhailova, A. G.; Румш, Л. Д.

doi:10.1385/abab:88:1-3:159

Cited by 12 publications

(15 citation statements)

References 20 publications

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“…As we revealed earlier, calcium ions are also required for the prevention of autolysis of enteropeptidase heavy chain, but the purification of this enzyme in the presence of CaCl 2 at all stages results in relatively stable preparations, whose autolysis requires an extensive incubation with EDTA or EGTA chelators [11]. Therefore, in those cases when the addition of CaCl 2 into substrate-enteropepti dase incubation mixture was undesired, we performed the hydrolysis without Ca 2+ .…”

Section: Resultsmentioning

confidence: 99%

“…Activity of enteropeptidase was determined by monitoring the increase in absorbance at 324 nm occurring upon hydro lysis of Z Lys S Bzl (66.7 µM) in the presence of 0. Enteropeptidase (EP 118 1035) was extracted from bovine duodenal mucosa and purified according to the technique developed previously [11]. Activity of the nat ural enzyme preparations was determined by trypsinogen activation [11].…”

Section: Methodsmentioning

confidence: 99%

“…Enteropeptidase (EP 118 1035) was extracted from bovine duodenal mucosa and purified according to the technique developed previously [11]. Activity of the nat ural enzyme preparations was determined by trypsinogen activation [11].…”

Section: Methodsmentioning

confidence: 99%

“…At the same time, the efficiency of GD 4 K Nfa hydrolysis by the light chain of the enzyme in the pres ence of 10 mM Ca 2+ is three times lower (due to higher K m value), and all constants virtually match the analogous ones for the case of full length human enzyme and low content of Ca 2+ (0.1 mM) [1] (Table 1). Now having two efficient synthetic substrates of enteropeptidase at our disposal, GD 4 K F(NO 2 )G and G 5 DK F(NO 2 )G, where F(NO 2 ) is a p nitrophenylala nine residue, as well as highly purified preparation of nat ural bovine enteropeptidase [11], preparation of a trun cated enteropeptidase, containing C terminal region of the heavy chain (466 800), and a recombinant form of the light chain of this enzyme, containing only the last 17 residues of the heavy chain (784 800), we have performed a systematic study of the effect of calcium ion on the hydrolysis of all the abovementioned substrates by these enteropeptidase forms.…”

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confidence: 99%

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Effect of Calcium Ions on Enteropeptidase Catalysis

Mikhailova

Likhareva

Прудченко

et al. 2005

Biochemistry (Moscow)

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The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Effect of Calcium Ions on Enteropeptidase Catalysis

Mikhailova

Likhareva

Прудченко

et al. 2005

Biochemistry (Moscow)

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“…(1a)). As a result of the systematic study of enzymatic and structural features of enteropeptidase, we have concluded that the unique properties of this highly specific and active enzyme are associated with the existence of the three substrate binding sites: S1 and S2 of the light chain, and, also, SII located in the region 118 -465 of the heavy chain [6][7][8].…”

Section: Introductionmentioning

confidence: 98%

The Ways of Realization of High Specificity and Efficiency of Enteropeptidase

Mikhailova¹,

Likhareva²,

Teich³

et al. 2007

PPL

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Comparative substrate analysis of full-length bovine enteropeptidase and trypsin, bovine and human enteropeptidase light chains was performed using model N-terminal dodecapeptides corresponding to wild-type human trypsinogen and pancreatitis-associated mutant trypsinogens K23R and D22G. The substitution of Lys residue by Arg at P1 leads to 2-fold increase in the efficiency of enteropeptidase hydrolysis; the absence of the negatively charged residue at P2 reduces the efficiency of such hydrolysis by two orders of magnitude. The difference in efficiency of peptide chain hydrolysis after Lys/Arg residues by enteropeptidase compared to trypsin is equal to the difference in hydrolysis by serine proteases of different primary specificity of their specific substrates.

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Proteins as supramolecular building blocks: Nterm‐Lsr2 as a new protein tecton

et al. 2015

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Proteins hold great promise in forming complex nanoscale structures which could be used in the development of new nanomaterials, devices, biosensors, electronics, and pharmaceuticals. The potential to produce nanomaterials from proteins is well supported by the numerous examples of self-assembling proteins found in nature. We have explored self-assembling proteins for use as supramolecular building blocks, or tectons, specifically the N-terminal domain of Lsr2, Nterm-Lsr2. A key feature of this protein is that it undergoes self-assembly via proteolytic cleavage, thereby allowing us to generate supramolecular assemblies in response to a specific trigger. Herein, we report the effects of pH and protein concentration on the oligomerization of Nterm-Lsr2. Furthermore, via protein engineering, we have introduced a new trigger for oligomerization via enteropeptidase cleavage. The new construct of Nterm-Lsr2 can be activated and assembled in a controlled fashion and provides some ability to alter the ratio of higher ordered structures formed.

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Enteropeptidase: Structure, Function, and Application in Biotechnology

Cited by 12 publications

References 20 publications

Effect of Calcium Ions on Enteropeptidase Catalysis

Effect of Calcium Ions on Enteropeptidase Catalysis

The Ways of Realization of High Specificity and Efficiency of Enteropeptidase

Proteins as supramolecular building blocks: Nterm‐Lsr2 as a new protein tecton

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