Hydrolysis Of Oligopeptides Research Articles

Mutations of key substrate binding residues of leishmanial peptidase T alter its functional and structural dynamics

BackgroundM20 aminopeptidases, such as Peptidase T (PepT), are implicated in the hydrolysis of oligopeptides during the terminal stages of protein degradation pathway to maintain turnover. Therefore, specific inhibition of PepT bores well for the development of novel next-generation antileishmanials. This work describes the metal dependence, substrate preferences and inhibition of PepT, and demonstrates in detail the role of its two conserved substrate binding residues. MethodsPepT was purified and characterized using a scheme of peptide substrates and peptidomimetic inhibitors. Residues T364 and N378 were mutated and characterized with an array of biochemical, biophysical and structural biology methods. ResultsPepT sequence carries conserved motifs typical of M20 peptidases and our work on its biochemistry shows that this cytosolic enzyme carries broad substrate specificity with best cleavage preference for peptides carrying alanine at the P1 position. Peptidomimetics amastatin and actinonin occupied S1 pocket by competing with the substrate for binding to active site and inhibited PepT potently, while arphamenine A and bestatin were less effective inhibitors. We further show that the mutation of conserved substrate binding residues (T364 and N378) to alanine affects structure, reduces substrate binding and alters the amidolytic activity of this dimeric enzyme. ConclusionsPepT preferentially hydrolyzes oligopeptides carrying alanine at P1 position and is potently inhibited by peptidomimetics. Reduced substrate binding after mutations was a key factor involved in amidolytic digressions. General significanceThis study provides insights for further exploration of the druggability of PepT and highlights prospective applications of this enzyme along with its mutazyme T364A/N378A.

Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese

We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than 3,000 small peptides, representing a fairly full casein peptidome containing peptides of 4 to 25 AA in length: 1,049 from β-casein (CN), 944 from αS1-CN, 813 from αS2-CN, and 234 from κ-CN. The majority of β-CN- and αS1-CN-derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from αS2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, whereas peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-CN, αS2-CN, as well as αS1-CN, showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β-CN and αS1-CN accumulated during cheese maturation. In contrast, we did not notice any generation of phosphopeptides from the highly phosphorylated part of αS2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin. The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free AA and reported the results in the paper.

Comparative Study of the Reactivity of Zirconium(IV)‐Substituted Polyoxometalates towards the Hydrolysis of Oligopeptides

AbstractThe hydrolytic activity of the ZrIV‐substituted Keggin‐type (Et2NH2)8[{α‐PW11O39Zr‐(μ‐OH)(H2O)}2]·7H2O (1), Lindqvist‐type (Me4N)2[W5O18Zr(H2O)3] (2), and Wells–Dawson‐type Na14[Zr4(P2W16O59)2(μ3‐O)2(OH)2(H2O)4]·57H2O (3) polyoxometalates (POMs) towards the peptide bonds in the oligopeptides triglycine, tetraglycine, glycylglycylhistidine, and glycylserylphenylalanine was investigated by kinetic methods and multinuclear NMR spectroscopy. 31P NMR and UV/Vis spectroscopy showed that 1–3 were stable under the conditions used to study peptide bond hydrolysis. The reactivity of 1–3 towards oligopeptides was compared on the basis of the amount of free glycine produced at a certain time increment. In the presence of 1–3, rate constants in the range 6.25 × 10–7 to 10.14 × 10–7 s–1 were obtained, whereas no hydrolysis was observed after one month in the absence of these POMs. The results showed that the Keggin‐type complex 1 was the most active towards peptide bond hydrolysis in tri‐ and tetrapeptides. 1H and 13C NMR spectroscopy showed that triglycine, tetraglycine, and glycylserylphenylalanine interact with 1 and 2 preferentially through the amine nitrogen atom and the N‐terminal amide oxygen atom to activate the peptide bond towards hydrolysis. The coordination of glycylglycylhistidine resulted in multiple complexes with 1–3 as a result of additional imidazole coordination to the ZrIV centers.

Anti‐integrase abzymes from the sera of HIV‐infected patients specifically hydrolyze integrase but nonspecifically cleave short oligopeptides

In contrast to canonical proteases, total immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies (Abs) from HIV-infected patients hydrolyze effectively only HIV integrase (IN), reverse transcriptase (RT), human casein, and serum albumin. Anti-IN IgG and IgM isolated by chromatography on IN-Sepharose hydrolyze specifically only IN but not many other tested proteins. Total Abs from HIV-infected patients hydrolyze not only globular proteins but also different specific and nonspecific tri-, tetra-, and 20- to 25-mer oligopeptides (OPs) with a higher rate than anti-IN Abs isolated using IN-Sepharose. A similar situation was observed for IgG from patients with multiple sclerosis and HIV-infected patients, which after purification on myelin basic protein (MBP)-Sepharose and RT-Sepharose specifically hydrolyze only MBP and RT, respectively. The active sites of all anti-protein abzymes are localized on their light chains, whereas the heavy chain is responsible for the affinity of protein substrates. Interactions of intact globular proteins with both light and heavy chains of abzymes provide the specificity of protein hydrolysis. The affinity of anti-IN and anti-MBP abzymes for intact IN and MBP is approximately 10(2)- to 10(5)-fold higher than for short and long specific and nonspecific OPs. The data suggest that all OPs interact mainly with the light chain of different Abs, which possesses a lower affinity for substrates, and therefore, depending on the OP sequences, their hydrolysis may be less specific or completely nonspecific. The data indicate that the relative activity of Abs not fractionated on specific protein sorbents in the hydrolysis of specific and nonspecific OPs can correspond to an average proteolytic activity of light chains of polyclonal Abs directed against many different proteins.

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 Ca(2+) 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.

Influence of Dietary Protein Level and Source on the Course of Protein Digestion Along the Small Intestine of the Veal Calf

The objective of this study was to examine the effect of the dietary crude protein (CP) content and source on the distribution of digesta proteins and peptides according to their molecular mass along the small intestine of veal calves. Diets contained 14, 104, 205, and 279 g/kg of CP supplied by skim milk powder (SMP) in experiment 1. Diets contained only SMP or SMP plus proteins (1:1 on digestible CP basis) from either a soybean protein concentrate (SPC), an isolated soy protein partially hydrolyzed (HSPI), or a potato protein concentrate (PPC) in experiment 2. Duodenal, jejunal, and ileal digesta were collected from calves fitted with simple cannulae and continuously infused the milk replacers into the abomasum. The distribution of molecular mass (Mr) of proteins and peptides was studied by gel filtration chromatography. Increasing the dietary CP level of milk replacers increased the flow of oligopeptides and free amino acids in ileal digesta. Incorporating plant protein increased the flow of proteins with Mr>20,000 in the duodenum and that of proteins and peptides with Mr<10,000 in the ileum. Hydrolysis of oligopeptides and absorption may be a limiting step in the digestion of plant protein in the veal calf.

Hydrolysis of oligopeptides by sera used in cell and tissue culture.

Sera commonly used in cell and tissue culture as medium supplements possess high peptidase activity. Pligopeptides incubated with 1% serum are rapidly hydrolyzed to intermediate length peptides and the constituent amino acids. Hydrolysis of lysine peptides is difficult to verify by a quantitative ninhydrin procedure because of reaction of the lysine epsi-lon-amino group and serum components with ninhydrin. Attempts to evaluate oligopeptides as sole sources of indispensable amino acids for cultured mammalian cells are of doubtful value when serum is used as a medium supplement.