A. G. Mikhailova

Autolysis of bovine enteropeptidase heavy chain: evidence of fragment 118^465 involvement in trypsinogen activation

Variations in bovine enteropeptidase (EP) activity were shown to result from autolysis caused by the loss of calcium ions; the cleavage sites were determined. The native enzyme preferred its natural substrate, trypsinogen (K M=2.4 μM), to the peptide and fusion protein substrates (K M=200 and 125 μM, respectively). On the other hand, the truncated enzyme composed of the C‐terminal fragment 466–800 of EP heavy chain and intact light chain did not distinguish these substrates. The results suggest that the N‐terminal fragment 118–465 of the enteropeptidase heavy chain contains a secondary substrate‐binding site that interacts directly with trypsinogen.

Psychrophilic trypsin-type protease from Serratia proteamaculans

A preparative method for purification of a novel protease from the psychrotolerant Gram-negative microorganism Serratia proteamaculans (PSP) was developed using affinity chromatography on BPTI-Sepharose. It yielded electrophoretically homogeneous PSP preparation of 60 kD. The PSP properties (temperature and pH stability, high catalytic efficiency) indicate that this enzyme can be defined as a psychrophilic protease. Inhibitory analysis together with substrate specificity indicates that the studied PSP exhibits properties of serine trypsin-like and Zn-dependent protease.

The Ways of Realization of High Specificity and Efficiency of Enteropeptidase

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.

Study of secondary specificity of enteropeptidase in comparison with trypsin.

A comparative study of secondary specificities of enteropeptidase and trypsin was performed using peptide substrates with general formula A-(Asp/Glu)n-Lys(Arg)-↓-B, where n = 1-4. This was the first study to demonstrate that, similar to other serine proteases, enteropeptidase has an extended secondary binding site interacting with 6-7 amino acid residues surrounding the peptide bond to be hydrolyzed. However, in the case of typical enteropeptidase substrates containing four negatively charged Asp/Glu residues at positions P2-P5, electrostatic interaction between these residues and the secondary site Lys99 of the enteropeptidase light chain is the main factor that determines hydrolysis efficiency. The secondary specificity of enteropeptidase differs from the secondary specificity of trypsin. The chromophoric synthetic enteropeptidase substrate G5DK-F(NO2)G (kcat/Km = 2380 mM–1·min–1) is more efficient than the fusion protein PrAD4K-P26 (kcat/Km = 1260 mM–1·min–1).

Effect of calcium ions on enteropeptidase catalysis.

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-α-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25°C were determined for natural enteropeptidase (Km 59.6 µM, kcat 6660 min−1-, kcat/Km 111 µM−1·min−1), as well as for enteropeptidase preparation with deleted 118–783 fragment of the heavy chain (Km 176.9 µM, kcat 6694 min−1, kcat/Km 37.84 µM−1·min−1) and trypsin (Km 56.0 µM, kcat 8280 min−1, kcat/Km 147.86 µ M−1·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 GD4K-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 GD4K-F(NO2)G, G5DK-F(NO2)G (where F(NO2) is p-nitrophenyl-L-phenylalanine residue), and GD4K-Nfa (where Nfa is β-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.

Enteropeptidase : Structure, function, and application in biotechnology

A preparative method for purification of enteropeptidase (enterokinase) (EC 3. 4. 21. 9) is developed. A highly purified form of this enzyme is stabilized by calcium ions and does not contain any other proteolytic enzyme contaminations. These enteropeptidase preparations were successfully used for cleavage of a variety of fusion proteins containing the tetraaspartyl-lysyl sequence in an arbitrary position on the polypeptide chain. A series of substrates was methodically studied, which resulted in the suggestion that the peptide and fusion protein substrates (K m = 200 μM and 125 μM, respectively) were bound to the enzyme through the linker (Asp) 4 Lys at the binding site on the light chain of enteropeptidase. Much more efficient hydrolysis of the natural substrate trypsinogen (K m = 2.4 μM) testifies to a significant contribution of other sites of the substrate and the enzyme in productive binding. A variation in the enzymes unique specificity was shown to be a result of the autolysis caused by the loss of calcium ions; the cleavage sites were determined. The truncated enzyme containing the C-terminal fragment 466-800 of its heavy chain and the intact light chain does not distinguish the natural substrate trypsinogen, fusion protein, or peptide substrates. These results suggest that the N-terminal fragment 118-465 of the enteropeptidase heavy chain contains a secondary substrate-binding site that interacts directly with trypsinogen.

Cloning, sequencing, expression, and characterization of thermostability of oligopeptidase B from Serratia proteamaculans, a novel psychrophilic protease

Protease from Serratia proteamaculans (PSP) is the first known psychrophilic oligopeptidase B. The gene of S. proteamaculans 94 oligopeptidase B was cloned, sequenced and expressed in Escherichia coli. The unfolding of PSP molecule following heat treatment at 37°C by measuring fluorescence spectra was examined in parallel with the residual activity determination. The effect of PSP thermostabilization by glycerol at 37-50 °С was revealed. Calcium ions and buffer solution of low molarity cause the opposite effect - the acceleration of PSP inactivation at 37°C. The thermal stability of PSP molecule in the presence of 0-100mM CaCl2 was also investigated by means of high-sensitivity differential scanning calorimetry. The artificial reconstruction of the natural complex PSP-chaperonin from S. рroteamaculans was carried out: the stable complex (1:1) of chaperonin E. сoli GroEL with active recombinant enzyme PSP was obtained. It was shown that complex formation with chaperonin promotes PSP thermostability at 37°C.

Oligopeptidase B from Serratia proteamaculans . I. Determination of primary structure, isolation, and purification of wild-type and recombinant enzyme variants

A novel trypsin-like protease (PSP) from the psychrotolerant gram-negative microorganism Serratia proteamaculans was purified by ion-exchange chromatography on Q-Sepharose and affinity chromatography on immobilized basic pancreatic trypsin inhibitor (BPTI-Sepharose). PSP formed a tight complex with GroEL chaperonin. A method for dissociating the GroEL-PSP complex was developed. Electrophoretically homogeneous PSP had molecular mass of 78 kDa; the N-terminal amino acid sequence 1–10 was determined, and mass-spectral analysis of PSP tryptic peptides was carried out. The enzyme was found to be the previously unknown oligopeptidase B (OpdB). The S. proteamaculans 94 OpdB gene was sequenced and the producer strain Escherichia coli BL-21(DE3) pOpdB No. 22 was constructed. The yield of expressed His6-PSP was 1.5 mg/g biomass.

Oligopeptidase B from Serratia proteamaculans. II. Enzymatic characteristics: Substrate analysis, influence of calcium ions, pH and temperature dependences

Enzymatic properties of a novel oligopeptidase B from psychrotolerant gram-negative microorganism Serratia proteamaculans (PSP) were studied. The substrate specificity of PSP was analyzed using p-nitroanilide substrates, and the influence of calcium ions on the enzyme activity was studied. Hydrolysis of oligopeptides by PSP was studied using melittin as the substrate. Optimal conditions for the PSP activity (pH and temperature) have been established. It was found that PSP shares some properties with oligopeptidases B from other sources containing two Asp/Glu residues in the S2 site, but it differs significantly in some characteristics. The S2 site of PSP contains only one Asp460 residue. The secondary specificity of PSP has a number of specific features: an unusual substrate inhibition by peptides with hydrophobic residues at the P2 position, as well as the drastic influence of calcium ions on substrate characteristics of the enzyme. It is assumed that the PSP molecule contains a large hydrophobic substrate-binding site, and significant conformational rearrangements of the enzyme active site are induced by Ca2+ binding and by the formation of the enzyme-substrate complex. The temperature characteristics of PSP (high activity at low temperature as well as low apparent temperature optimum (25°C)) confirm that PSP is a psychrophilic enzyme.

Synthetic model and bioactive peptides as potential substrates for enteropeptidase

Enteropeptidase (enterokinase EC 3.4.21.9), catalyzing trypsinogen activation, exhibits unique properties for high efficiency hydrolysis of the polypeptide chain after the N-terminal tetraaspartyl-lysyl sequence. This makes it a convenient tool for the processing of fusion proteins containing this sequence. We found the enteropeptidase-catalysing degradation of some bioactive peptides: cattle hemoglobin beta-chain fragments Hb (2–8) (LTAEEKA) and Hb (1–9) (MLTAEEKAA), human angiotensin II (DRVYIHPF) (AT). Model peptides with truncated linker WDDRG and WDDKG also were shown to be susceptible to enteropeptidase action. Kinetic parameters of enteropeptidase hydrolysis for these substrates were determined.K m values for all substrates with truncated linker (≈10−3 M) are an order of magnitude higher than corresponding values for typical enteropeptidase artificial peptide or fusion protein substrates with full enteropeptidase linker-DDDDK-(K m ≈10−4 M).k cat values for AT, Hb (2–8), WDDRG and WDDKG are ≈30–40 min−1. But one additional amino acid residue at both N-and C-terminus of Hb (2–8) results in a drastic increase of hydrolysis efficiency:k cat value for Hb (1–9) is 1510 min−1. Recent study demonstrates the possibility of undesirable cleavage of target peptides or proteins containing the above-mentioned truncated linker sequences; further, the ability of enteropeptidase to hydrolyse specifically several biologically active peptidesin vitro along with its unique natural substrate trypsinogen was demonstrated.