Cryoenzymology of porcine pepsin

Physical and kinetic properties of porcine pepsin have been examined in aqueous methanol solvents at temperatures below ambient to seek evidence for covalent intermediates in the catalyzed hydrolysis of good substrates. It was first demonstrated that aqueous methanol cryosolvents have no significant deleterious effects upon this protein. The addition of methanol does lead to a drastic reduction in the midpoint of the thermal melting curve of pepsin. This could account for rate reductions previously observed in catalysis by this enzyme. This effect is lessened by the addition of active-site ligands including substrates and is fully reversible upon dilution into aqueous solution. Two substrates were chosen which have chromophoric groups on opposite sides of the scissile peptide bond. The UV spectral changes from hydrolysis of Pro-Thr-Glu-Phe-(NO2)Phe-Arg-Leu and the fluorescence spectral changes from hydrolysis of DNS-Ala-Ala-Phe-Phe-OP4P+-CH3 were studied at temperatures down to -60 degrees C. The resulting Arrhenius plots were linear in the region where pepsin exists in the native state with downward curvature exhibited at higher temperatures where the reversible denaturation occurs. No "burst" reactions were observed with either substrate. In addition, efforts at trapping intermediates by low-temperature denaturation and precipitation have provided no evidence for covalent intermediates on the reaction pathway. Although this evidence is negative, we cannot rule out the possibility of the formation of covalent intermediates following an initial rate-limiting step.     

Anticoagulative effect of pepsin

Anticoagulative effect of pepsin is observed in vitro when its concentration is 36 microM and higher. This effect is due to inhibition of fibrin monomer polymerization. Protamine abolishes anticoagulative effect of pepsin. Pepsin does not influence platelet aggregation induced by ADP and collagen.     

Structural basis for the inhibition of porcine pepsin by Ascaris pepsin inhibitor-3

The three-dimensional structures of pepsin inhibitor-3 (PI-3) from Ascaris suum and of the complex between PI-3 and porcine pepsin at 1. 75 A and 2.45 A resolution, respectively, have revealed the mechanism of aspartic protease inhibition by this unique inhibitor. PI-3 has a new fold consisting of two domains, each comprising an antiparallel beta-sheet flanked by an alpha-helix. In the enzyme-inhibitor complex, the N-terminal beta-strand of PI-3 pairs with one strand of the 'active site flap' (residues 70-82) of pepsin, thus forming an eight-stranded beta-sheet that spans the two proteins. PI-3 has a novel mode of inhibition, using its N-terminal residues to occupy and therefore block the first three binding pockets in pepsin for substrate residues C-terminal to the scissile bond (S1'-S3'). The molecular structure of the pepsin-PI-3 complex suggests new avenues for the rational design of proteinaceous aspartic proteinase inhibitors.     

Structural dissection of alkaline-denatured pepsin

It has been established in a number of studies that the alkaline-denatured state of pepsin (the I(P) state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I(P) state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I(P) state of pepsin. CD spectra indicate in addition that significant nonnative alpha-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I(P) state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe.     

Ultraviolet difference spectra of pepsin

A shift of pH of pepsin solutions from 4.6 to 1.0 gives rise to spectral displacements in the ultraviolet. If represented as difference spectra three peaks with maxima at 2770, 2850, and 2930 Ångströms are present which can be attributed to the tyrosine and tryptophan residues in the protein. On mild autolysis of pepsin at pH 2.0 the absorbancy in the ultraviolet further decreases. Although some of these effects can be ascribed to the occurrence of hydrogen bonding between the aromatic residues and a carboxylate ion, those observed on autolysis are caused by charge effects of newly formed polar groups in the vicinity of a chromophore. No direct relation between the optical properties described here and enzymic activity of pepsin has been observed.     

Multiple forms of horse pepsin

Using ion-exchange and affinity chromatography and isoelectrofocusing, eight forms of pepsin with pI 1.6, 1.8, 2.1, 2.3, 2.6, 2.8, 3.2 and 3.6, were isolated from horse gastric juice. The molecular weights, amino acid composition, N-terminal sequence and functional activity of these multiple forms were determined. Partial primary structure of tryptic peptides of pepsin with pI 2.3 was investigated. The analyzed partial sequences of the forms with pI 1.8, 2.1, 2.3, and 2.6 have identical structures which differ from the amino acid sequence of pepsin with pI 3.2 by four substituents. In terms of their functional activity, horse pepsins differ only insignificantly. Presumably, the pepsins under study (at least the forms with pI 1.8, 2.1, 2.3, 2.6 and 3.2) arose comparatively recently as a result of duplication of the common precursor gene and exist at an early stage of structural and functional divergence. As far as their primary structure and functional properties are concerned, these pepsins are more related to pepsin A than to other isoenzymes of gastric aspartyl proteinases of mammalia, e. g., gastricsin or chymosin.