Obtaining higher transesterification rates with subtilisin Carlsberg in nonaqueous media.

Three phase partitioning (protein precipitate obtained as an interfacial layer between lower aqueous and upper t-butanol phases, formed by the addition of ammonium sulphate and t-butanol to the aqueous solution of protein) followed by lyophilization in the presence of two-component excipient resulted in 400-480x increases in transesterification activity of lyophilized powders of subtilisin Carlsberg, depending on the solvent. The three phase partitioned enzyme, 'dried' by washing with butanol, gave 3-4x higher rates (depending on the solvent used) than the enzyme preparation dried by lyophilization in the presence of two-component excipient system.

Proprotein convertases regulate activity of prostate epithelial cell differentiation markers and are modulated in human prostate cancer cells.

Prostate derived factor (PDF) is a member of transforming growth factor-beta (TGF-beta) superfamily proteins involved in differentiation of the prostate epithelium. Proprotein convertases (PCs) such as furin are thought to mediate the processing of TGF-beta superfamily. In the present study, we demonstrated for the first time that human prostate cancer cell lines differentially synthesize and secret prostate derived factor (PDF), and that PDF secreted by LNCaP is processed by PCs. Exposure of LNCaP cells to the decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK), a synthetic furin-like protease inhibitor, inhibited PDF processing and resulted in the loss of luminal cell phenotype and induction of basal cell phenotype in LNCaP cells as demonstrated by alternations in the expression of cytokeratins 8, 14, 18, and 19, markers of prostate epithelial cell differentiation. These results suggest that proprotein convertases may be involved in the regulation of prostate epithelial cell differentiation, and may be an important target of prostate cancer therapy.

Enzyme activation in organic solvents: co-lyophilization of subtilisin Carlsberg with methyl-beta-cyclodextrin renders an enzyme catalyst more active than the cross-linked enzyme crystals.

In this study we explored the efficiency of the additive methyl-beta-cyclodextrin (M beta CD) to enhance the activity and enantioselectivity of the serine protease subtilisin Carlsberg in organic solvents. These two parameters, measured for different transesterification reactions and in several solvents, are compared with results obtained by using two additional preparations of the same enzyme: lyophilized powder and cross-linked enzyme crystals (CLEC). The results suggest that co-lyophilization of subtilisin with M beta CD preserves the enzyme's active site tertiary structure rendering a highly active and enantioselective catalyst.

Peptide synthesis in organic media with subtilisin 72 immobilized on poly(vinyl alcohol)-cryogel carrier.

Serine proteinase subtilisin 72 was covalently attached to the beads of poly(vinyl alcohol)-cryogel, a macroporous hydrogel prepared by the freeze-thaw technique. The immobilized enzyme was examined as a catalyst in the synthesis of protected peptides Z-Ala-Ala-Xaa-Phe-pNA (Xaa = Leu, Glu, Lys) in acetonitrile/dimethylformamide mixtures. Immobilized subtilisin catalyzed with high yield the formation of peptide bonds between Phe-pNA and acyl donors including those with free carboxylic group and non-protected C-terminal basic and acidic amino acid residues.

Activation of enzymes for nonaqueous biocatalysis by denaturing concentrations of urea.

Urea is one of the most commonly used denaturants of proteins. However, herein we report that enzymes lyophilized from denaturing concentrations of aqueous urea exhibited much higher activity in organic solvents than their native counterparts. Thus, instead of causing deactivation, urea effected unexpected activation of enzymes suspended in organic media. Activation of subtilisin Carlsberg (SC) in the organic solvents (hexane, tetrahydrofuran, and acetone) increased with increasing urea concentrations up to 8 M. Active-site titration results and activity assays indicated the presence of partially unfolded but catalytically active SC in 8 M urea; however, the urea-modified enzyme retained high enantioselectivity and was ca. 80 times more active than the native enzyme in anhydrous hexane. Likewise, the activity of horseradish peroxidase (HRP) lyophilized from 8 M urea was ca. 56 times and 350 times higher in 97% acetone and water-saturated hexane, respectively, than the activity of HRP lyophilized from aqueous buffer. Compared with the native enzyme, the partially unfolded enzyme may have a more pliant and less rigid conformation in organic solvents, thus enabling it to retain higher catalytic activity. However, no substantial activation was observed for alpha-chymotrypsin lyophilized from urea solutions in which the enzyme retained some activity, illustrating that the activation effect is not completely general.

Effect of intramolecular cross-linking between glutamine-41 and lysine-50 on actin structure and function.

Subdomain 2 of actin is a dynamic segment of the molecule. The cross-linking of Gln-41 on subdomain 2 to Cys-374 on an adjacent monomer in F-actin inhibits actomyosin motility and force generation (Kim et al., 1998; Biochemistry 37, 17,801-17,809). To shed light on this effect, additional modifications of the Gln-41 site on actin were carried out. Both intact G-actin and G-actin cleaved by subtilisin between Met-47 and Gly-48 in the DNase 1 binding loop of subdomain 2 were treated with bacterial transglutaminase. According to the results of Edman degradation, transglutaminase introduced an intramolecular zero-length cross-linking between Gln-41 and Lys-50 in both intact and subtilisin cleaved actins. This cross-linking perturbs G-actin structure as shown by the inhibition of subtilisin and tryptic cleavage in subdomain 2, an allosteric inhibition of tryptic cleavage at the C-terminus and decrease of modification rate of Cys-374. The cross-linking increases while the subtilisin cleavage dramatically decreases the thermostability of F-actin. The Mg- and S1-induced polymerizations of both intact and subtilisin cleaved actins were only slightly influenced by the cross-linking. The activation of S1 ATPase by actin and the sliding speeds of actin filaments in the in vitro motility assays were essentially unchanged by the cross-linking. Thus, although intramolecular cross-linking between Gln-41 and Lys-50 perturbs the structure of the actin monomer, it has only a small effect on actin polymerization and its interaction with myosin. These results suggest that the new cross-linking does not alter the intermonomer interface in F-actin and that changes in actomyosin motility reported for the Gln-41-Cys-374 intrastrand cross-linked actin are not due to decreased flexibility of loop 38-52 but to constrains introduced into the F-actin structure and/or to perturbations at the actin's C-terminus.

The prosegments of furin and PC7 as potent inhibitors of proprotein convertases. In vitro and ex vivo assessment of their efficacy and selectivity.

All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme. In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160. These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases. Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition. The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs. These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor. Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

Optimizing the salt-induced activation of enzymes in organic solvents: effects of lyophilization time and water content.

The addition of simple inorganic salts to aqueous enzyme solutions prior to lyophilization results in a dramatic activation of the dried powder in organic media relative to enzyme with no added salt. Activation of both the serine protease subtilisin Carlsberg and lipase from Mucor javanicus resulting from lyophilization in the presence of KCl was highly sensitive to the lyophilization time and water content of the sample. Specifically, for a preparation containing 98% (w/w) KCl, 1% (w/w) phosphate buffer, and 1% (w/w) enzyme, varying the lyophilization time showed a direct correlation between water content and activity up to an optimum, beyond which the activity decreased with increasing lyophilization time. The catalytic efficiency in hexane varied as much as 13-fold for subtilisin Carlsberg and 11-fold for lipase depending on the lyophilization time. This dependence was apparently a consequence of including the salt, as a similar result was not observed for the enzyme freeze-dried without KCl. In the case of subtilisin Carlsberg, the salt-induced optimum value of kcat/Km for transesterification in hexane was over 20,000-fold higher than that for salt-free enzyme, a substantial improvement over the previously reported enhancement of 3750-fold (Khmelnitsky, 1994). As was found previously for pure enzyme, the salt-activated enzyme exhibited greatest activity when lyophilized from a solution of pH equal to the pH for optimal activity in water. The active-site content of the lyophilized enzyme samples also depended upon lyophilization time and inclusion of salt, with opposite trends in this dependence observed for the solvents hexane and tetrahydrofuran. Finally, substrate selectivity experiments suggested that mechanism(s) other than selective partitioning of substrate into the enzyme-salt matrix are responsible for salt-induced activation of enzymes in organic solvents.

Proprotein-processing endoprotease furin controls growth of pancreatic beta-cells.

We have previously reported that in the well-differentiated beta-cell line MIN6 cells, the beta-cell-specific differentiated characteristics, such as insulin content, expression of prohormone convertases PC2 and PC3, and glucose-regulated insulin secretion, diminished when the proprotein-processing endoprotease furin was highly expressed. Since furin converts many growth-related protein precursors to their bioactive forms, we compared the four pancreatic islet cell lines RINm5F, betaTC3, betaHC9, and MIN6 with respect to cell growth rate, furin expression, endoprotease activity, and insulin content. RINm5F cells exhibited the strongest furin expression, higher furin-type endoprotease activity, and the fastest cell growth, but had the least insulin content. In contrast, MIN6 cells exhibited only a weak furin expression, little furin-type endoprotease activity, and the slowest cell growth, but had the highest insulin content. To test whether furin-expressing cells secrete growth-promoting factors cleaved by furin, we prepared conditioned media from RINm5F and furin cDNA-introduced MIN6 (MIN6-F) cells. The conditioned media from RINm5F and MIN6-F induced increased DNA synthesis and promoted the growth of normal MIN6 cells, compared with the medium from the empty vector-introduced MIN6-0 cells. We then examined the effect of the protease inhibitors alpha1-antitrypsin and its variants by infecting their vaccinia recombinants to the four cell lines. All conditioned media from each cell line expressing the furin-specific alpha1-antitrypsin variant exhibited the least DNA synthetic capacity on normal MIN6 cells. Furthermore, all three sublines of MIN6-F grew faster than MIN6-0 and MIN6. Thus, we suggest that the islet cells with higher furin expression may induce increased production of growth factors, which result in an increase in cell growth, through an autocrine/paracrine mechanism.

A molecular dynamics study of the effect of carbon tetrachloride on enzyme structure and dynamics: subtilisin.

Developing enzymes that are functional in highly concentrated halocarbon solutions, such as carbon tetrachloride, may prove useful in the development of new strategies for environmental remediation and monitoring of pollutant plumes, as well as in developing 'green' processes. Doing so will require gaining an understanding of the underlying structural and dynamic effects on enzymes induced by such solvents. Herein we report a 714 ps molecular dynamics simulation of the enzyme subtilisin Carlsberg and its waters of crystallization in a periodic box of carbon tetrachloride. The crystal structure from aqueous solution was used as the starting structure for our simulation using the AMBER program and forcefield. The calculated time-averaged structure is similar to the aqueous X-ray structure except for significant differences in loop (or turn) regions, resulting in many extra intra-protein hydrogen bonding interactions. Since carbon tetrachloride is a non-polar solvent and cannot interact strongly with the protein and water molecules, the water molecules stay very close to the protein surface throughout the simulation. The mobility of most of the waters was therefore very low. A few water molecules underwent significant lateral motion during the simulation, but never wandered far from the protein surface. Waters were either hydrogen bonded to protein polar groups, other water and/or counterions. Some of the surface waters participated in the formation of water-mediated hydrogen bonding networks. The increase in total number of intra-protein hydrogen bonds and the formation of water-mediated hydrogen bonding networks in carbon tetrachloride is consistent with the generally observed increase in thermostability and reduced flexibility of proteins in non-aqueous solutions. Several possible carbon tetrachloride binding sites on the protein surface are predicted.

Achievement of renaturation of subtilisin BPN' by a novel procedure using organic salts and a digestible mutant of Streptomyces subtilisin inhibitor.

The pro-sequences of proteases have been considered to be required for the refolding of denatured proteases. However, here we report achievement of almost complete restoration of enzymatic activity of subtilisin BPN' in the absence of its pro-sequence. The presence of 2 M potassium acetate in the folding medium enhanced the refolding efficiency of guanidine hydrochloride (GdnHCl)-denatured subtilisin BPN' by up to 28%, and other organic salts were also found to be useful, suggesting that general contribution of the bulky hydrophobic moieties of the salts to the formation of a favorable environment required for folding. This finding will provide new insights into the folding mechanisms not only of proteases but also of various other proteins. Almost complete restoration of enzymatic activity of denatured subtilisin in the organic salt solution was accomplished by further addition of mutated Streptomyces subtilisin inhibitor (SSI), which had been converted to a digestible temporary inhibitor by removal of the disulfide bridge near the reactive site.

The elastolytic properties of subtilisin GX from alkalophilic Bacillus sp. strain 6644 provides a means of differentiation from other subtilisins.

A serine protease exhibiting high activity in alkaline media was purified from alkalophilic Bacillus strain GX6644. The enzyme, subtilisin GX, has a molecular weight of 25,000 and a pI greater than 9.5. The protease exhibited high elastolytic activity, but unlike most elastin hydrolyzing enzymes, elastin hydrolysis and binding were not inhibited by 0.1 M NaCl. The elastolytic properties of subtilisin GX together with its specificity toward amino acid phenyl esters functionally distinguishes this protease from other subtilisins. However, comparisons of the available amino-terminal sequence of subtilisin GX with subtilisins from alkalophilic and neutrophilic Bacillus species revealed extensive homology.

Effects of deletion in the flexible loop of the protease inhibitor SSI (Streptomyces subtilisin inhibitor) on interactions with proteases.

The Streptomyces subtilisin inhibitor (SSI) is a proteinaceous protease inhibitor which inhibits serine proteases by forming a stable Michaelis complex. The flexible loop region (Thr64-Val69) is a very flexible region in an SSI molecule and its importance in interactions with proteases has been suggested, since conformational change of this loop was found to occur for the smooth binding of SSI with various proteases. In this study, mutated SSIs lacking one or two residues in this region were generated and the effects of deletions on the interaction with proteases were investigated. Deletion was introduced into mutated SSI(Lys73) and SSI(Gly70Lys73) both known to be trypsin inhibitors, to examine the effects of deletion on interactions with subtilisin BPN' or trypsin. The deletion of one residue (Gly66) caused increased inhibitory activity toward trypsin, indicating the protruding flexible loop hinders binding with trypsin. Reduction of such hindrance by one-residue shortening in this loop is shown to be effective for the interaction of SSI(Lys73) with trypsin. In contrast, one-residue shortening had virtually no effect on inhibition toward subtilisin BPN'. Differences in the subsite structures of these proteases may have been the reason for this contrast. The deletion of two residues (Thr64 and Gly66) in this region converted SSI into a temporary inhibitor. Structural analysis of the degradation intermediate showed that the peptide bond at the reactive site of doubly deleted SSI was cleaved by subtilisin BPN' after its binding with protease. Thus, the irreversibility of the cleaved peptide bond at the reactive site of mutated SSI in the complex with protease may possibly be the cause for its temporary inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Spectroscopic studies on proteinase K and subtilisin DY. Relation to X-ray models.

Circular dichroic spectroscopy has been used to study the effect of pH, guanidinium hydrochloride concentration and temperature on the conformation of the fungal subtilisin-like proteinase K and the bacterial DY. The ellipticity of the bands in the far ultraviolet region remains almost unchanged in the pH range 3.0-11.0 (PMS-proteinase K) and 5.0-10.0 (PMS-subtilisin DY). The same ranges of pH stability were determined from the pH dependence of the near ultraviolet dichroic spectra. Hence the changes in the tertiary and secondary structure occur in parallel. Proteinase K is considerably more stable at acidic and somewhat more stable at alkaline pH than subtilisin DY. At neutral pH proteinase K is more resistant to denaturation by guanidinium hydrochloride than is subtilisin DY. The midpoints of the denaturation curves were 6.2 M and 3.2 M guanidinium, respectively. The thermal unfolding of proteinase K occurred at a higher temperature than for subtilisin DY, the transition midpoints being 65 degrees and 48 degrees, respectively. Thus proteinase K is overall a much more robust molecule than subtilisin DY, showing greater resistance to all three forms of denaturation. The differences in the stability of the two proteinases can be partly explained by differences in their calcium binding sites.

[The inhibition of proteolytic enzyme activity by humic acids]. / Ingibirovanie aktivnosti proteoliticheskikh fermentov guminovymi kislotami.

The inhibitory effect of peat humic acids on the hydrolysis of N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-leucine methyl ester by alpha-chymotrypsin and subtilism has been studied. Samples of humic acids with M(W) approximately 18,000 have been used in experiments. The results of kinetic studies indicates the mixed type of inhibition of proteinase activity by humic acids. The meanings of inhibition constants under the action of humic acids on alpha-chymotrypsin and subtilism have been calculated.