Processive action of the two peptide binding sites of prolyl 4-hydroxylase in the hydroxylation of procollagen

The number of peptide binding sites of prolyl 4-hydroxylase was manipulated with the peptide photoaffinity label N-(4-azido-2-nitrophenyl)glycyl-(Pro-Pro-Gly)5, and the effect on hydroxylation of the relatively short peptide substrate (Pro-Pro-Gly)5 and of the long natural substrate procollagen was studied. With (Pro-Pro-Gly)5 as a substrate, a linear relation was found between enzyme activity and the amount of covalently bound photoaffinity label, approximately 50% inactivation being reached at 1 mol of label/mol of enzyme. No difference in Km value for (Pro-Pro-Gly)5 was detected between unlabeled and partially labeled enzyme preparations. These results indicate that enzyme molecules with only one free active site hydroxylated the synthetic substrate (Pro-Pro-Gly)5 with the same Km and at half the rate of native enzyme. In contrast, with procollagen as a substrate a 5-10-fold increase in Km was found with the fraction of enzyme containing only one free active site, as compared to the Km for procollagen with nonlabeled enzyme. This finding is explained by an enzyme-kinetic model based on a processive action of the two peptide substrate binding sites of prolyl 4-hydroxylase, preventing dissociation of the enzyme-substrate complex between successive hydroxylations of a long peptide with multiple substrate sites. Such a mechanism leads to a low Km for a long peptide by overcoming the diffusional constraints on the rate of association between the enzyme and the individual substrate sites.     

Human lipoprotein lipase: the loop covering the catalytic site is essential for interaction with lipid substrates.

Lipoprotein lipase (LPL), a key enzyme which initiates the hydrolysis of triglycerides present in chylomicrons and very low density lipoproteins, consists of multiple functional domains which are necessary for normal activity. The catalytic domain of LPL mediates the esterase function of the enzyme but separate lipid binding sites have been proposed to be involved in the interaction of LPL with emulsified lipid substrates at the water-lipid interface. Like pancreatic lipase (PL), LPL contains a surface loop covering the catalytic pocket that may modulate access of the substrate to the active site of the enzyme. Secondary structural analysis of this loop reveals a helix-turn-helix motif with two short amphipathic helices that have hydrophobic moments of 0.64 and 0.68. In order to investigate the role of the loop in the initial interaction of LPL with its substrate, we utilized site-directed mutagenesis to generate eight constructs in which the amphipathic properties of the loop were altered and expressed them in human embryonal kidney-293 cells. Reducing the amphiphilicity without changing the predicted secondary structure of the loop abolished the ability of the lipase to hydrolyze emulsified, long chain fatty acid triglycerides (triolein) but not the water soluble substrate tributyrin. Replacing the loop of LPL with the loop of hepatic lipase, which differs in 15 of 22 amino acids but is also amphiphilic, led to the expression of an enzyme that retained both triolein and tributyrin hydrolyzing activity. Substitution of the LPL loop by a short four amino acid peptide, which may allow more direct access to the active site than the 22 amino acid loop, enhanced hydrolysis of short chain fatty acid triglycerides by more than 2-fold, while the ability to hydrolyze emulsified substrates was abolished. Thus, disruption of the amphipathic structure of the LPL loop selectively decreases the hydrolysis of emulsified lipid substrate without affecting the esterase or catalytic function of the enzyme. These studies establish that the loop with its two amphipathic helices is essential for hydrolysis of long chain fatty acid substrate by LPL providing new insight into the role of the LPL loop in lipid-substrate interactions. We propose that the interaction between the lipoprotein substrates and the amphipathic helices within this loop may in part determine lipase substrate specificity.     

Proline-specific endopeptidases from microbial sources: isolation of an enzyme from a Xanthomonas sp.

An extensive screening among microorganisms for the presence of post-proline-specific endopeptidase activity was performed. This activity was found among ordinary bacteria from soil samples but not among fungi and actinomycetes. This result is in contrast to the previous notion that this activity is confined to the genus Flavobacterium. A proline endopeptidase was isolated from a Xanthomonas sp. and characterized with respect to physicochemical and enzymatic properties. The enzyme is composed of a single peptide chain with a molecular weight of 75,000. The isoelectric point is 6.2. It is inhibited by diisopropylfluorophosphate and may therefore be classified as a serine endopeptidase. The activity profile is bell shaped with an optimum at pH 7.5. By using synthetic peptide substrates and intramolecular fluorescence quenching it was possible to study the influence of substrate structure on the rate of hydrolysis. The enzyme specifically hydrolyzed Pro-X peptide bonds. With Glu at position X, low rates of hydrolysis were observed; otherwise the enzyme exhibited little preference for particular amino acid residues at position X. A similar substrate preference was observed with respect to the amino acid residue preceding the prolyl residue in the substrate. The enzyme required a minimum of two amino acid residues toward the N terminus from the scissile bond, but further elongation of the peptide chain by up to six amino acid residues caused only a threefold increase in the rate of hydrolysis. Attempts to cleave at the prolyl residues in oxidized RNase failed, indicating that the enzyme does not hydrolyze long peptides, a peculiar property it shares with other proline-specific endopeptidases.     

Detection of human neutrophil elastase with peptide-bound cross-linked ethoxylate acrylate resin analogs.

An assessment of elastase-substrate kinetics and adsorption at the solid-liquid interface of peptide-bound resin was made in an approach to the solid-phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N-succinyl-alanine-alanine-proline-valine-p-nitroanilide (suc-Ala-Ala-Pro-Val-pNA), a chromogenic HNE substrate, was attached to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. To assess the enzyme-substrate reaction in a two-phase system, the kinetic profile of resin-bound peptide substrate hydrolysis by HNE was obtained. A glycine and di-glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc-Ala-Ala-Pro-Val-pNA and suc-Ala-Ala-Pro-Ala-pNA on the solid-phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate-resin and enzyme concentration. Enzyme hydrolysis of the resin-bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin-released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate-elastase adsorption and activity at the resin's solid-liquid interface for HNE detection with a solid-phase peptide.     

Inhibitory effect of a self-derived peptide on glucosyltransferase of Streptococcus mutans. Possible novel anticaries measures.

Glucosyltransferase (GTF) plays an important role in the development of dental caries. We examined the possible presence of self-inhibitory segments within the enzyme molecule for the purpose of developing anticaries measures through GTF inhibition. Twenty-two synthetic peptides derived from various regions presumably responsible for insoluble-glucan synthesis were studied with respect to their effects on catalytic activity. One of them, which is identical in amino acid sequence to residues 1176-1194, significantly and specifically inhibited both sucrose hydrolysis and glucosyl transfer to glucan by GTF-I. Double-reciprocal analysis revealed that the inhibition is noncompetitive. Scramble peptides, composed of the identical amino acids in randomized sequence, had no effect on GTF-I activity. Furthermore, the peptide is tightly bound to the enzyme once complexed, even in the presence of sodium dodecyl sulfate (SDS). Kinetic analysis using an optical evanescent resonant mirror cuvette system demonstrated that the enzyme-peptide interaction was biphasic. These results indicate that the peptide directly interacts with the enzyme with high affinity and inhibits its activity in a sequence-specific manner. This peptide itself could possibly be an effective agent for prevention of dental caries, although its effectiveness may be improved by further modification.     

Effects of subzero temperatures on the kinetics of protease catalyzed dipeptide synthesis in organic media

A depeptide synthesis was drastically influenced by the reaction temperature, in the range from -30 degrees to 25 degrees C. This article shows the kinetic reasons of this effect. alpha-Chymotrypsin was immobilized on celite and used in four different water-miscible solvents containing small amounts of water-miscible solvents containing small amounts of water. The reaction studied was the aminolysis of N-acetyl-L-phenylalanine ethyl ester (Ac-PheOEt) with L-alaninamide (Ala-NH(2)) and water for the acylenzyme complex, the nucleophile was favoured by low reaction temperatures. This effect (quantified as p-values) was observed in all four solvents, and it was greatest in acetonitrile and tetrahydrofuran. The esterase and amidase activities of the enzyme were studies using AcPheOEt and N-acetyl-L-phenylalanyl-L-ananinamide (AcPheAla-NH(2)) as substrates. The Michaelis-Menten parameters, K(m,app) and V(max), were determined for ester hydrolysis and dipeptide hydrolysis. Both K(m,app) and V(max) tended to increase with increasing temperature. Secondary hydrolysis was reduced at subzero temperatures because ester hydrolysis was favoured in relation to depeptide hydrolysis. Depeptide synthesis was thus favored by low temperatures in two ways: first, in the competition between the nucleophile and water for the acyl enzyme; and, second, in the competition between the ester substrate and the peptide substrate for the free enzyme. As a result, in acetonitrile containing 10% water, the maximal yield was 99% at -20%C compared with 84% at 25 degrees C. (c) 1995 John Wiley & Sons, Inc.     

Crystal structure and enhanced activity of a cutinase‐like enzyme from Cryptococcus sp. strain S‐2

The structural and enzymatic characteristics of a cutinase‐like enzyme (CLE) from Cryptococcus sp. strain S‐2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 Å resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long‐chain substrates, as well as the preferred specificity of FS cutinase for short‐chain substrates. In addition, site‐directed mutagenesis was performed to increase the hydrolysis activity on long‐chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long‐chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.     

Immobilisation procedure and reaction conditions for optimal performance of Candida antarctica lipase B in transesterification and hydrolysis

Abstract

The reaction kinetics of Candida antarctica lipase B (CalB) in the commercially available preparation Novozym^® 435 (N435) were compared to those of preparations of CalB immobilised on Accurel^® MP1000 (porous polypropylene). Two polypropylene preparations were made using enzyme loadings of 0.2% and 2% (w/w). All three preparations were used in hydrolysis as well as transesterification of two substrates, ethyl acrylate and ethyl methacrylate with octanol. Reactions carried out at water activity levels from 0.06 to 0.96 and at octanol concentrations between 25 and 500 mM showed that both water and octanol can inhibit CalB. Pronounced mass transfer limitations were also observed, which were more pronounced for N435 than for the two MP1000 preparations. The MP1000 preparations could thus use the lipase more efficiently in these reactions, achieving a specific activity (per g enzyme) between 5 and 20 times that of N435. To achieve high rates in the transesterification reaction, it is recommended to use low water activity and moderate alcohol concentration. In order to carry out a hydrolysis reaction, an intermediate water activity should be used to balance the effects of water as a limiting substrate and as a competitive inhibitor.     

Regulation of sphingomyelin long chain base synthesis in human fibroblasts in culture. Role of lipoproteins and the low density lipoprotein receptor

We have studied regulation of synthesis of long chain bases in human fibroblasts using 3 different radioactive precursors and 2 different hydrolysis and separation procedures. Serum and low density lipoproteins inhibited synthesis. Inhibition of long chain base synthesis by various concentrations of low density lipoproteins paralleled inhibition of cholesterol synthesis. This inhibition was dependent on the low density lipoprotein receptor pathway since fibroblasts from homozygous familial hypercholesterolemic patients did not show the inhibition observed with normal fibroblasts. Incorporation of precursor palmitate into free or total long chain bases was inhibited by low density lipoproteins to the same extent as incorporation into sphingomyelin long chain bases. We thus propose that an enzyme in the pathway leading to sphinganine synthesis, probably palmitoyl-CoA:L-serine C-palmitoyltransferase (decarboxylating) EC 2.3.1.50, is regulated by low density lipoproteins.     

Specificity of Proteinase K from Tritirachium album Limber for Synthetic Peptides

The specificity of proteinase K from Tritirachium album Limber was determined using various synthetic peptide substrates. The esterase activity against N-acylated amino acid esters indicated that the enzyme is primarily specific against aromatic or hydrophobic amino acid residues at the carboxyl side of the splitting point. Secondary interaction for hydrolysis was also studied using peptide esters or others, which showed that the enzyme activity is markedly promoted by elongating the peptide chain to the N-terminal from the splitting point. Thus, peptide chloromethyl ketone derivatives such as Cbz-Ala-Gly-PheCH₂Cl inactivated the enzyme activity markedly.     

Design, synthesis and catalytic activity of a serine protease synthetic model

The design, synthesis and catalytic properties of acyclic branched peptide carrier that possesses thecatalytic triad residues of the serine proteases isreported. The synthesis of the peptide model wastotally completed on solid support using threedifferent orthogonal amino protecting groups.Hydrolytic activity measurements againstSuc-Ala-Ala-Ala-pNA substrate showed that it ishydrolysed by the peptide model to a small extent.Despite this small hydrolytic activity, it is thefirst time, to our knowledge, that hydrolysis of such a substrate is reported by an enzyme model compound.Contrary, this enzyme model peptide showedconsiderable activity against the Boc-Ala-pNPsubstrate (k_cat = 0.414 min^–1 and K_m = 0.228 mm). These results suggest that thedesigned carrier brings in appropriate contact thecatalytic triad residues (Ser, His, Asp) resulting inthe obtained hydrolytic activity.     

Protease La, the lon gene product, cleaves specific fluorogenic peptides in an ATP-dependent reaction

Protease La is an ATP-dependent protease that catalyzes the rapid degradation of abnormal proteins and certain normal polypeptides in Escherichia coli. In order to learn more about its specificity and the role of ATP, we tested whether small fluorogenic peptides might serve as substrates. In the presence of ATP and Mg2+, protease La hydrolyzes two oligopeptides that are also substrates for chymotrypsin, glutaryl-Ala-Ala-Phe-methoxynaphthylamine (MNA) and succinyl-Phe-Leu-Phe-MNA. Methylation or removal of the acidic blocking group prevented hydrolysis. Closely related peptides (glutaryl-Gly-Gly-Phe-MNA and glutaryl-Ala-Ala-Ala-MNA) are cleaved only slightly, and substrates of trypsin-like proteases are not hydrolyzed. Furthermore, several peptide chloromethyl ketone derivatives that inhibit chymotrypsin and cathepsin G (especially benzyloxycarbonyl-Gly-Leu-Phe-chloro-methyl ketone), inhibited protease La. Thus its active site prefers peptides containing large hydrophobic residues, and amino acids beyond the cleavage site influence rates of hydrolysis. Peptide hydrolysis resembles protein breakdown by protease La in many respects: 1) ADP inhibits this process rapidly, 2) DNA stimulates it, 3) heparin, diisopropyl fluorophosphate, and benzoyl-Arg-Gly-Phe-Phe-Leu-MNA inhibit hydrolysis, 4) the reaction is maximal at pH 9.0-9.5, 5) the protein purified from lon- E. coli or Salmonella typhymurium showed no activity against the peptide, and that from lonR9 inhibited peptide hydrolysis by the wild-type enzyme. With partially purified enzyme, peptide hydrolysis was completely dependent on ATP. The pure protease hydrolyzed the peptide slowly when only Mg2+, Ca2+, or Mn2+ were present, and ATP enhanced this activity 6-15-fold (Km = 3 microM). Since these peptides cannot undergo phosphorylation, adenylylation, modification of amino groups, or denaturation, these mechanisms cannot account for the stimulation by ATP. Most likely, ATP and Mg2+ affect the conformation of the enzyme, rather than that of the substrate.     

The mechanism of biosynthesis and direction of chain extension of a polyl-(N-acetylglucosamine 1-phosphate) from the walls of Staphylococcus lactis N.C.T.C. 2102

1. The synthesis of a polymer of N-acetylglucosamine 1-phosphate, occurring in the walls of Staphylococcus lactis N.C.T.C. 2102, was examined by using cell-free enzyme preparations. The enzyme system was particulate, and probably represents fragmented cytoplasmic membrane. 2. Uridine diphosphate N-acetylglucosamine was the only substrate required for polymer synthesis and labelled substrate was used to show that N-acetylglucosamine 1-phosphate is transferred as an intact unit from substrate to polymer. 3. The properties of the enzyme system were studied. A high concentration of Mg(2+) or Mn(2+) was required for optimum activity, and the pH optimum was about 8.5. 4. End-group analysis during synthesis in vitro showed that newly formed chains contain up to about 15 repeating units. Pulse-labelling indicated that chain extension occurs by transfer from the nucleotide to the ;sugar-end' of the chain, i.e. to the end that is not attached to peptidoglycan in the wall.     

Influence of solvent and water activity on kinetically controlled peptide synthesis.

alpha-Chymotrypsin deposited on Celite was used to catalyse peptide synthesis reactions between N-protected amino acid esters and leucine amide in organic media with low water content. The influence of the solvent and the thermodynamic water activity on the reaction kinetics was studied. The substrate specificity in the reactions was shown to be a combination of the substrate specificity of the enzyme in aqueous media and the influence of the solvents. The magnitude of the solvent effects differed greatly depending on the substrates used. In hydrophobic solvents high reaction rates were observed and the competing hydrolysis of the ester substrate occurred to only a minor extent. Reactions occurred at water activities as low as 0.11, but the rate constants increased with increasing water activity and were about two orders of magnitude higher at the highest water activity tested (0.97).     

Interactions of alpha-chymotrypsin with peptides containing tryptophan or its derivatives at the C-terminus.

The interaction between alpha-chymotrypsin [EC 3.4.21.1] and peptide substrate or peptide inhibitor was investigated to determine how the secondary interaction influences the rate of hydrolysis or the binding and whether or not its effect is variable with alteration of the P1 residue which interacts with the specificity determining site of the enzyme. Kinetic analysis was carried out at pH 6.5 and 7.8 for substrates of the type Ac-Glyn-X-OMe and for inhibitors of the type Ac-Glyn-X-OH where X denotes tryptophan or its derivatives. With substrates containing tryptophan or Nin-formyltryptophan, the second-order rate of hydrolysis increases with increase of chain length. With substrates containing 2-(2-nitro-4-carboxyphenylsulfenyl)-tryptophan, however, the rate of hydrolysis decreases with elongation of the chain, due to an increase in Km(app). The corresponding inhibitors behave differently from the other series of inhibitors at pH 6.5. The results indicate that the influence of the secondary interaction on reactivity or binding is related to the structural features of the P1 residue.     

Rat hepatic microsomal acetoacetyl-CoA reductase. A beta-ketoacyl-CoA reductase distinct from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system

The present study provides evidence for a new rat liver microsomal enzyme, a short chain beta-ketoacyl (acetoacetyl)-CoA reductase, which is separate from the long chain beta-ketoacyl-CoA reductase component of the microsomal fatty acid chain elongation system. This microsomal reductase converts acetoacetyl-CoA to beta-hydroxybutyryl-CoA at a rate of 70 nmol/min/mg of protein; the enzyme has a specific requirement for NADH and appears to obtain electrons directly from the reduced pyridine nucleotide without the intervention of cytochrome b5 and its flavoprotein reductase. The apparent Km of the enzyme of the acetoacetyl-CoA was 21 microM and for the cofactor, 18 microM. The pH optimum was broad, ranging from 6.5 to 8.0. The product formed is the D-isomer of beta-hydroxybutyryl-CoA. High carbohydrate fat-free diet resulted in a small but significant (35%) increase in microsomal acetoacetyl-CoA reductase activity. The cytosol also contains this enzyme activity, measuring approximately 57% of that found in the microsomes. The mitochondrial activity which is 20-25% higher than the microsomal activity appears to be due to L-beta-hydroxyacyl-CoA dehydrogenase which converts acetoacetyl-CoA to L-beta-hydroxybutyryl-CoA. The microsomal acetoacetyl-CoA reductase activity was extracted from the microsomal membrane by 0.4 M KCl, resulting in an 8- to 10-fold purification; in addition, the long chain fatty acid elongation system was unaffected by this extraction procedure. Employing beta- hydroxyhexanoyl -CoA as a substrate, evidence is also provided for a separate dehydratase which acts on short chain substrates. Lastly, the liver microsomes had no detectable acetoacetyl-CoA synthetase or acetyl-CoA acetyltransferase activities. Hence, the possible involvement of the rat hepatic microsomal short chain beta-ketoacyl-CoA reductase, short chain beta-hydroxyacyl-CoA dehydratase, and the previously reported short chain trans-2-enoyl-CoA reductase in the hepatic utilization of acetoacetyl-CoA and in the synthesis of butyryl-CoA for hepatic lipogenesis is discussed.     

The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Modulation of protease specificity by a change in the enzyme microenvironment. Selectivity modification on a model substrate, purified soluble proteins and gluten

Subtilisin BPN' activity on a synthetic substrate is found to decrease with the concentration of soluble additives such as sugars and polyols, the catalytic efficiency of the enzyme being related to the water activity in the reaction medium. Limited hydrolysis of B chain of insulin is followed and the cleavage priority determined. When carried out in glycerol-containing medium, both enzyme catalytic behaviour and specificity are perturbed; a different cleavage order and a selectivity restriction are observed. The experiments were generalised to purified proteins and to an insoluble protein complex. The hydrolysis kinetics of purified gliadins by pepsin and of gluten by a Bacillus neutral protease are modulated in presence of water activity depressors. Glycerol is able to increase both pepsin efficiency and gluten protein solubility. The hydrolysis order is affected by water-structuring molecules in the enzyme microenvironment and new peptides appear whatever the size and initial solubility of the substrate.     

Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitin-conjugating enzyme

Protein modification with lysine 63-linked ubiquitin chains has been implicated in the non-proteolytic regulation of signaling pathways. To understand the molecular mechanisms underlying this process, we have developed an in vitro system to examine the activity of the ubiquitin-conjugating enzyme UBC13-UEV1A with TRAF6 in which TRAF6 serves as both a ubiquitin ligase and substrate for modification. Although TRAF6 potently stimulates the activity of UBC13-UEV1A to synthesize ubiquitin chains, it is not appreciably ubiquitinated. We have determined that the presentation of Lys(63) of ubiquitin by UEV1A suppresses TRAF6 modification. Based on our observations, we propose that the modification of proteins with Lys(63)-linked ubiquitin chains occurs through a UEV1A-independent substrate modification and UEV1A-dependent Lys(63)-linked ubiquitin chain synthesis mechanism.