Effect of end group blockage on the properties of a class A amphipathic helical peptide

In a recent classification of biologically active amphipathic α-helixes, the lipid-associating domains in exchangeable plasma apolipoproteins have been classified as class A amphipathic helixes (Segrest, J. P., De Loof, H., Dohlman, J. G., Brouillette, C. G., Anantharamaiah, G. M. Proteins 8:103–117, 1990). A model peptide analog with the sequence, Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (18A), possesses the characteristics of a class A amphipathic helix. The addition of an acetyl group at the α-amino terminus and an amide at the α-carboxyl terminus, to obtain Ac-18A-NH₂, produces large increases in helicity for the peptide both in solution and when associated with lipid (for 18A vs Ac-18A-NH₂, from 6 to 38% helix in buffer and from 49 to 92% helix when bound to dimyristoyl phosphatidylcholine in discoidal complexes). Blocking of the end-groups of 18A stabilizes the α-helix in the presence of lipid by approximately 1.3 kcal/mol. There is also an increase in the self-association of the blocked peptide in aqueous solution. The free energy of binding to the PC–water interface is increased only by about 3% (from ?8.0 kcal/mol for 18A to ?8.3 kcal/mol for Ac-18A-NH₂). The Ac-18A-NH₂ has a much greater potency in raising the bilayer to hexagonal phase transition temperature of dipalmitoleoyl phosphatidylethanolamine than does 18A. In this regard Ac-18A-NH₂ more closely resembles the behavior of the apolipoprotein A-I, which is the major protein component of high-density lipoprotein and a potent inhibitor of lipid hexagonal phase formation. The activation of the plasma enzyme lecithin: cholesterol acyltransferase by the Ac-18A-NH₂ peptide is greater than the 18A analog and comparable to that observed with the apo A-I. In the case of Ac-18A-NH₂, the higher activating potency may be due, at least in part, to the ability of the peptide to micellize egg PC vesicles. © 1993 Wiley-Liss, Inc.     

Development of helix-stabilized antimicrobial peptides composed of lysine and hydrophobic α,α-disubstituted α-amino acid residues

Lysine-based amphipathic nonapeptides, including homochiral peptides [Ac-(l-Lys-l-Lys-Xaa)₃-NH₂ (Xaa = Gly, Ala, Aib, Ac₅c, or Ac₆c) and Ac-(d-Lys-d-Lys-Aib)₃-NH₂], a heterochiral peptide [Ac-(l-Lys-d-Lys-Aib)₃-NH₂], and a racemic mixture of diastereomeric peptides [Ac-(rac-Lys-rac-Lys-Aib)₃-NH₂] were designed and synthesized to investigate the relationship between their preferred secondary structures and their antimicrobial activity. Peptide 5, [Ac-(l-Lys-l-Lys-Ac₆c)₃-NH₂] formed a stable α-helical structure and exhibited strong activity against Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa).     

Purification and some properties of elastase from hepatopancreas of king crab Paralithodes camtschatica

• 1.1. Elastase has been purified from the hepatopancreas of the king crab (Paralithodes camtschatica). Specific activity of the enzyme measured toward Suc-(Ala)₃-pNA and Boc-(Ala)₃-pNA was 926 and 3700 mUnits per mg of protein, respectively.
• 2.2. The enzyme is an anion protein (pI 4.5) with an approximate mol.wt of 28.5 kDa.
• 3.3. The enzyme exhibited a bell-shaped pH-dependence for the hydrolysis of Suc-(Ala)₃-pNA with a maximum at 8–8.5. Under these conditions the values of K_m and k_cat of the crab elastase are 4 mM and 4.75 s⁻¹, respectively.
• 4.4. The serine elastase is effectively inhibited by elastinal and diisopropylfluorophosphate.
• 5.5. It is shown that some salts except HgCl₂ activate the protease. In the presence of HgCl₂ with concentrations of 10 mM and higher, the crab elastase is inactive. SDS and Triton X-100 have no any effect on the activity of crab elastase.

     

N-acetyl-(L-Ala) 3 -p-nitroanilide as a new chromogenic substrate for elastase

The introduction of a useful new chromogenic substrate for the determination of elastase (EC 3.4.4.7) activity is described. N-acetyl-L-Ala-L-Ala-L-Ala-$\text{p}$-nitroanilide (AcAla₃NA) is a new specific elastase substrate whose hydrolysis can be followed spectrophotometrically at 410 nm in a wide pH range. Its rate of hydrolysis by α-chymotrypsin (EC 3.4.4.5) and trypsin (EC 3.4.4.4.) is 0.02% and 0.001% respectively compared to its rate of hydrolysis by elastase. As little as 0.1 μg elastase/ml can be satisfactorily determined. At pH 8, K_m = 0.88 mM and k_cat = 11.9 sec^?1.     

Synthesis and optical studies of L-methionine oligopeptides in solution

A series of L -methionine oligomers [BOC-(Met)_n-OMe] (n = 2–7) and the corresponding diastereomeric di- and tripeptides were synthesized using the mixed anhydride method. Oligomers prepared in this manner were optically pure and were obtained in reasonable yield. Preliminary optical examination of the peptides suggests that secondary structures may begin forming in the pentamer or hexamer in trifluoroethanol. BOC-(Met)₄-OMe and BOC-(Met)₅-OMe were also synthesized using an insoluble resin containing BOC-L -methionine as the nitrophenol active ester.     

Designing of peptides with left handed helical structure by incorporating the unusual amino acids

The conformational behaviour of delta Ala has been investigated by quantum mechanical method PCILO in the model dipeptide Ac-delta Ala-NHMe and in the model tripeptides Ac-X-delta Ala-NHMe with X = Gly, Ala, Val, Leu, Abu and Phe and is found to be quite different. The computational results suggest that in the model tripeptides the most stable conformation corresponds to phi 1 = -30 degrees, psi 1 = 120 degrees and phi 2 = psi 2 = 30 degrees in which the > C = 0 of the acetyl group is involved in hydrogen bond formation with N-H of the amide group. Similar results were obtained for the conformational behaviour of D-Ala in Ac-D-Ala-NHMe and Ac-Ala-D-Ala-NHMe. The conformational behaviour of the amino acids delta Ala, D-Ala, Val and Aib in model tripeptides have been utilized in the designing of left handed helical peptides. It is shown that the peptide HCO-(Ala-D-Ala)3-NHMe can adopt both left and right handed helix whereas in the peptide Ac-(Ala-delta Ala)3-NHMe the lowest energy conformer is beta-bend ribbon structure. Left handed helical structure with phi = 30 degrees, psi = 60 degrees for D-Ala residues and phi = psi = 30 degrees for delta Ala is found to be more stable by 4 kcal mole-1 than the corresponding right handed helical structure for the peptide Ac-(D-Ala-delta Ala)3-NHMe. In both the peptides Ac-(Val-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe the most stable conformer is the left handed helix. Comparisons of results for Ac-(Ala-delta Ala)3-NHMe and Ac(Val-delta Ala)3-NHMe and Ac-(D-Ala-delta Ala)3-NHMe and Ac-(D-Val-delta Ala)3-NHMe also reveal that the Val residues facilitate the population of 3(10) left handed helix over the other conformers. It is also shown that the conformational behaviour of Aib residue depends on the chirality of neighbouring amino acids, i.e. Ac-(Aib-Ala)3-NHMe adopts right handed helical structure whereas Ac-(Aib-D-Ala)3-NHMe is found to be in left handed helical structure.     

Secondary interactions in mesentericopeptidase-catalyzed hydrolysis of peptide ester and 4-nitroanilide substrates

Five substrate series with the formulae Z-(Gly)n-Phe-OMe, Z-(Ala)n-Phe-OMe, Ac-(Ala)n-Phe-OMe, Z-(Gly)n-Phe-NA, and Suc-(Gly)n-Phe-NA (n = 0-2) (Z-benzyloxycarbonyl) were synthesized and used to study the active site of mesentericopeptidase (EC 3.4.21). The elongation of the peptide chain in all series leads to a 100- to 300-fold increase of kcat/Km. This indicates an extended substrate binding site, comprising at least three subsites (S1-S3). The sequence P1-P3 that fits these subsites is Phe-Ala-Ala. Mesentericopeptidase responds to the elongation of the peptide chain in the series Ac-(Ala)n-Phe-OMe in a way similar, but not identical, to subtilisin Carlsberg and subtilisin BPN'. The poor amidolytic activity of mesentericopeptidase and subtilisins toward 4-nitroanilides with peptide sequences matching the S1-S3 subsites is discussed in terms of unfavorable S'1-P'1 interaction.     

Alkaline serine proteinases D and E of Streptomyces griseus K-1.

Two DFP-sensitive alkaline proteinases with strong esterase activity toward Ac-(Ala)3-OMe, designated as alkaline serine proteinases D and E, were purified pronase, a protease mixture from St. griseus K-1. Each was shown to be homogeneous by acrylamide disc gel electrophoresis. The molecular weights of these enzymes were estimated to be about 27,000 be gel filtration. Studies on their actions on acyl-tl-amino acid methyl or ethyl esters indicated that proteinases D and E both exhibited a broad substrate specificity and hydrolyzed the ester bonds of esters containing Trp, Tyr, Phe, Leu, and Ala. The esterase activities of both enzymes toward Ac-(Ala)3-OMe were the highest among proteinases so far isolated from various sources. Proteinases D and E also lacked cystine residues in their molecules, being entirely different from alkaline serine proteinases A, B, and C in pronase. Some differences were , however, observed between them as regards pH stability, behavior on CM-cellulose, mobility on polyacrylamide electrophoresis, and amidase activity toward Suc-(Ala)3-pNA.     

Detection of elastase activity with a zymogram method after isoelectric focusing in polyacrylamide gel

A zymogram method for detecting elastase activity following isoelectric focusing in polyacrylamide gel is described. After enzyme activity has been visualized, the gel itself is available for protein staining and for analysis in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in second dimension. The zymogram method is suitable for detecting microgram amounts of elastase and has one step only. It can be used with the purified enzyme as well as with crude extracts of tissue containing elastases showing activity toward succinyl-(Ala)₃-p-nitroanilide. By this method a major component of elastase in both porcine and rat pancreas was detected. In addition, two forms of elastase with isoelectric points of 8.2 and 8.8, respectively, were identified in rat leukocyte extracts.     

Sensitive substrates for human leukocyte and porcine pancreatic elastase: a study of the merits of various chromophoric and fluorogenic leaving groups in assays for serine proteases.

Five sensitive substrates of human leukocyte and porcine pancreatic elastase having the sequence MeO-Suc-Ala-Ala-Pro-Val-X where -X is -NA (4-nitroanilide), -SBzl (thiobenzyl ester), -OEt, -AMC (4-methyl-7-coumarylamide), or -NNapOMe (1-methoxy-3-naphthylamide), were synthesized. The kinetic constants for the enzymatic hydrolysis as well as the sensitivity of each substrate are reported. Hydrolysis of the peptide -AMC and -NNapOMe derivatives were followed by monitoring spectrofluorometrically the release of H-AMC and H-NNapOMe, respectively. Cleavage of the thiobenzyl ester yields benzyl mercaptan as the hydrolysis product. Its release was monitored at 412 nm by reaction with Ellman's reagent [5,5′-dithiobis(2-nitrobenzoic acid)] in the assay mixture to produce the 3-carboxy-4-nitrothiophenoxide anion or at 324 nm by reaction with 4,4′-dithiodipyridine to produce 4-thiopyridone. Hydrolysis of the ethyl ester was measured using a coupled assay with NAD⁺ and liver alcohol dehydrogenase. The NADH⁺ formed upon oxidation of the ethanol released from cleavage of the ester was followed at 340 nm. MeO-Suc-Ala-Ala-Pro-Val-SBzl, the best substrate of the series, was capable of detecting as little as 2.4 pm (0.072 ng/ml) of active-site titrated human leukocyte elastase and 5.8 pm (0.15 ng/ml) of active-site titrated porcine pancreatic elastase using 4,4′-dithiodipyridine. The corresponding values with Ellman's reagent were 5.0 pm (0.15 ng/ml) and 7.4 pm (0.19 ng/ml), respectively. Advantages of this substrate are its high $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and ease of synthesis. One disadvantage is the interference of high concentration of thiols with the assay. The peptidyl-AMC is almost as sensitive as the thiobenzyl ester and can detect 11 pm of HL elastase and 18 pm of PP elastase. An advantage of this substrate is the fact that cleavage involves a peptide bond. Disadvantages are relatively low $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$ values and greater difficulty in synthesis. The peptidyl-NNapOMe has possible utility in histochemical studies due to the low intrinsic fluorescence of the substrate relative to the peptidyl-AMC. For a rate assay it has a lower sensitivity than either the thiobenzyl ester or peptidyl-AMC. The coupled liver alcohol dehydrogenase-ethyl ester assay offers no advantages except in cases where the ester substrate is commercially available. The 4-nitroanilide assay enjoys moderate sensitivity and is extremely convenient for routine use. Except for the peptidyl ethyl ester assay, all of the human leukocyte elastase assays reported in this paper are vastly more sensitive than any other existing assay for this protease.     

An elastolytic proteinase from rabbit leukocytes: purification and partial characterization

A proteinase with elastolytic activity was isolated from granules of rabbit bloodstream leukocytes, and purified to apparent homogeneity by a multi-step procedure consisting of ammonium sulfate precipitation, batch fractionation on DEAE-Sephadex A-50, and finally by preparative isoelectric focusing (IEF) on Sephadex G-75 Superfine. The molecule weight of the enzyme, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 28,500. This enzyme shows an isoelectric point at pH 9.0. The proteinase is active against natural elastins as well as toward Suc-(Ala)3-NA, Methoxy-Suc-(Ala)2-Pro-Val-NA, and (to a lesser extent) against Suc-(Ala)2-Pro-Leu-NA and Boc-Ala-ONp. The inhibition profile of the isolated enzyme indicates that rabbit granulocyte elastase belongs to the group of serine proteinases. Inhibition by some natural proteinase inhibitors is also observed. Unlike other mammalian elastases, it is insensitive to elastatinal.     

Estimation of the circular dichroism exhibited by statistical coils of poly(L-alanine) and unionized poly(L-lysine) in water

The circular dichroism of Ac-(Ala)_x-OMe and H-Lys-(Lys)_x-OH with x = 1, 2, 3, and 4 has been measured in aqueous solutions. The oligomers with x = 4 show similar circular dichroism spectra in water when the lysyl amino groups are protonated, and they respond in similar fashion to heating and to sodium perchlorate. Both oligomers at 15°C exhibit a positive circular dichroism band at 217–218 nm, which is eliminated by the isothermal addition of 4 M sodium perchlorate or by heating. The positive circular dichroism of the lysine oligomer is also eliminated when the pH is elevated to deprotonate the amino groups. Positive circular dichroism is still observed for Ac-(Ala)₄-OMe at elevated pH. Circular dichroism spectra have been estimated for poly(L -alanine) and poly(L -lysine) as statistical coils under the above conditions, based on the trends established with the oligomers. Poly(L -lysine) and poly(L -alanine) are predicted to exhibit similar circular dichroism behavior in aqueous solution so long as the lysyl amino groups are protonated. The circular dichroism of the statistical coil of poly(L -lysine), but not poly(L -alanine), is predicted to change when the pH is elevated sufficiently to deprotonate the lysyl amino groups. These results suggest that the unionized lysyl side chains participate in interactions that are not available to poly(L -alanine). Hydrophobic interactions may occur between the unionized lysyl side chains. Protonation of the lysyl amino groups is proposed to disrupt these interactions, causing poly(L -alanine) and protonated poly(L -lysine) to have similar circular dichroism properties.     

Characterization of the esterase isozymes of Ips typographus (coleoptera,scolytidae)

Four esterase isozymes hydrolyzing α-naphthyl acetate (α-NA) were detected screening whole body homogenates of larvae and adults of Ips typographus by electrophoresis. Two of the four isozymes (isozymes 3 and 4) were not detected by α-NA staining in the pupal stage, but topical application of juvenile hormone III (JH III) on the pupa induced these isozymes. The JH esterase (JHE) activity on the gel was associated with the proteins of isozyme 2. The compounds OTFP, PTFP, and DFP inhibited this catalytic activity of isozyme 2 on the gel at low concentrations, whereas the proteins of isozyme 3 and 4 were affected only at higher concentrations. A quantitative developmental study was performed to characterize which of the esterases hydrolyzed JH III, using a putative surrogate substrate for JH (HEXTAT) and α-NA. The I₅₀ of several esterase inhibitors and the JH metabolites were also defined. All findings supported the results that a protein associated with isozyme 2 is catabolizing JH and that isozymes 3 and 4 are the main contributors to the general esterase activity on α-NA. The JHE from Tenebrio molitor was purified by affinity chromatography. Although the recovery was low, an analytical isoelectric focusing gel showed that the JHE activity of the purified enzyme. T. molitor cochromatographed at the same pl as the JHE activity of I. typographus. Arch. Insect Biochem. Physiol. 34:203–221, 1997. © 1997 Wiley-Liss, Inc.     

Computer Modelling Studies on the Mechanism of Action of Ribonuclease T1

Abstract

The mechanism of action of ribonuclease (RNase) T₁ is still a matter of considerable debate as the results of x-ray, 2-D nmr and site-directed mutagenesis studies disagree regarding the role of the catalytically important residues. Hence computer modelling studies were carried out by energy minimisation of the complexes of RNase T₁ and some of its mutants (His40Ala, His40Lys, and Glu58Ala) with the substrate guanyl cytosine (GpC), and of native RNase T₁ with the reaction intermediate guanosine 2′, 3′-cyclic phosphate (G>p). The puckering of the guanosine ribose moiety in the minimum energy conformer of the RNase T₁ - GpC (substrate) complex was found to be O4′-endo and not C3′-endo as in the RNase T₁ - 3′-guanylic acid (inhibitor/product) complex. A possible scheme for the mechanism of action of RNase T₁ has been proposed on the basis of the arrangement of the catalytically important amino acid residues His40, Glu58, Arg77, and His92 around the guanosine ribose and the phosphate moiety in the RNase T₁ - GpC and RNase T₁ - G>p complexes. In this scheme, Glu58 serves as the general base group and His92 as the general acid group in the transphosphorylation step. His40 may be essential for stabilising the negatively charged phosphate moiety in the enzyme-transition state complex.     

Thermodynamics and mechanism of alpha helix initiation in alanine and valine peptides

We used molecular dynamics simulations to study the folding/unfolding of one of turn of an alpha helix in Ac-(Ala)3-NHMe and Ac-(Val)3-NHMe. Using specialized sampling techniques, we computed free energy surfaces as functions of a conformational coordinate that corresponds to alpha helices at small values and to extended conformations at large values. Analysis of the peptide conformations populated during the simulations showed that alpha helices, reverse turns, and extended conformations correspond to minima on the free energy surfaces of both peptides. The free energy difference between alpha helix and extended conformations, determined from the equilibrium constants for helix unfolding, is approximately -1 kcal/mol for Ac-(Ala)3-NHMe and -5 kcal/mol for Ac-(Val)3-NHMe. The mechanism observed in our simulations, which includes reverse turns as important intermediates along the helix folding/unfolding pathway, is consistent with a mechanism proposed previously. Our results predict that both peptides (but especially the Ala peptide) have a much larger equilibrium constant for helix initiation than is predicted by the helix-coil transition theory with the host-guest parameters. We also predict a much greater difference in the equilibrium constants than the theory predicts. Insofar as helix initiation is concerned, our results suggest that the large difference between the helical propensities of Ala and Val cannot be explained by simple concepts such as side-chain rotamer restriction or unfavorable steric interactions. Rather, the origin of the difference appears to be quite complicated because it involves subtle differences in the solvation of the two peptides. The two peptides have similar turn-extended equilibria but very different helix-turn equilibria, and the difference in helical propensities reflects the fact that the helix-turn equilibrium strongly favors the turns in Ac-(Val)3-NHMe, while it favors the helices in Ac-(Ala)3-NHMe. We also computed thermodynamic decompositions of the free energy surfaces, and these revealed that the helix-turn equilibria are vastly different primarily because the changes in peptide-water interactions that accompany helix-to-turn conformational changes are qualitatively different for the two peptides.     

A Comprehensive Alanine-Scanning Mutagenesis Study Reveals Roles for Salt Bridges in the Structure and Activity of Pseudomonas aeruginosa Elastase

The relationship between salt bridges and stability/enzymatic activity is unclear. We studied this relationship by systematic alanine-scanning mutation analysis using the typical M4 family metalloprotease Pseudomonas aeruginosa elastase (PAE, also known as pseudolysin) as a model. Structural analysis revealed seven salt bridges in the PAE structure. We constructed ten mutants for six salt bridges. Among these mutants, six (Asp189Ala, Arg179Ala, Asp201Ala, Arg205Ala, Arg245Ala and Glu249Ala) were active and four (Asp168Ala, Arg198Ala, Arg253Ala, and Arg279Ala) were inactive. Five mutants were purified, and their catalytic efficiencies (k _cat/K _m), half-lives (t _1/2) and thermal unfolding curves were compared with those of PAE. Mutants Asp189Ala and Arg179Ala both showed decreased thermal stabilities and increased activities, suggesting that the salt bridge Asp189-Arg179 stabilizes the protein at the expense of catalytic efficiency. In contrast, mutants Asp201Ala and Arg205Ala both showed slightly increased thermal stability and slightly decreased activity, suggesting that the salt bridge Asp201-Arg205 destabilizes the protein. Mutant Glu249Ala is related to a C-terminal salt bridge network and showed both decreased thermal stability and decreased activity. Furthermore, Glu249Ala showed a thermal unfolding curve with three discernable states [the native state (N), the partially unfolded state (I) and the unfolded state (U)]. In comparison, there were only two discernable states (N and U) in the thermal unfolding curve of PAE. These results suggest that Glu249 is important for catalytic efficiency, stability and unfolding cooperativity. This study represents a systematic mutational analyses of salt bridges in the model metalloprotease PAE and provides important insights into the structure-function relationship of enzymes.     

Variations in elastaselike esterase activities in human leucocytes during cell maturation.

Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development.     

Tuning the substrate selectivity of meta-cleavage product hydrolase by domain swapping

meta-Cleavage product (MCP) hydrolases can catalyze relatively low reactive carbon–carbon bond hydrolysis of products, which are derived from the meta-cleavage of catechols. The strict substrate selectivity of MCP hydrolases attracts an interest to understand the determinants of substrate specificity. Compared with conventional site-directed mutagenesis, domain swapping is an effective strategy to explore substrate specificity due to the large-scale reorganization of three-dimensional structure. In the present study, the hybrid MCP hydrolases BphD_LidA and MfphA_LidD were constructed by exchanging the lid domain of two parental enzymes MfphA and BphD. The residues Gly130/Ala196 (MfphA) and Gly136/Ala211 (BphD) were selected as crossover points according to structural disruption score analysis and molecular dynamics simulations. It was shown that the hybrid enzymes exhibited similar substrate selectivity with the parent enzyme providing the lid domain. Docking studies suggested that the lid domain may play a key role in determining substrate specificity by reshaping the active pocket and modulating the orientation of the substrate.     

Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Isolation and characterisation of two chymotrypsins from the midgut of Locusta migratoria

Two chymotrypsin isozymes (CTR 1 and CTR 2) from the midgut lumen of Locusta migratoria have been identified and purified. MALDI-TOF mass spectrometry revealed an M_r of 22 679 (±30) for CTR 1 and 22 592 (±30) for CTR 2. Both chymotrypsins hydrolysed S-(Ala)₂ProPhe-pNA (CTR 1: K_m=0.29±0.01 mM, V_max=83.0±1.4 U/mg; CTR 2: K_m=0.42±0.01 mM, V_max=48.9±1.1 U/mg) and S-(Ala)₂ProLeu-pNA (CTR 1: K_m=0.50±0.04 mM, V_max=1.7±0.1 U/mg; CTR 2: K_m=1.12±0.08 mM, V_max=11.4±0.6 U/mg), but neither enzyme hydrolysed BTpNA, S-Phe-pNA, Ac-Leu-pNA or S-(Ala)₃-pNA. CTR 1 and CTR 2 activities were effectively inhibited by AEBSF, PMSF, TPCK, chymostatin, SBTI and BPTI. Using S-(Ala)₂ProPhe-pNA as the substrate, CTR 1 gave optimal activity between pH 8.0 and 10.0, while CTR 2 was optimally active over the range pH 8.0–11.0. The N-terminal 15 amino acids of the purified chymotrypsins were determined, revealing their unique sequences which are also different from another, previously characterised Locusta chymotrypsin.