Time-dependent Structure and Activity Changes of α-Chymotrypsin in Water/Alcohol Mixed Solvents

Secondary structure of α-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. α-Chymotrypsin formed β-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, α-chymotrypsin took on a native-like structure, which gradually changed to β conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, α-chymotrypsin tended to form stable β-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to α-helix structure with substantial loss in catalytic activity was observed when α-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50%. In water/2,2,2-trifluoroethanol 90/10, α-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of β-sheet structure.     

Implications of the chemical modification of chymotrypsin

The short N-terminal chain of δ-chymotrypsin, a two-chain member of the chymotrypsin family, was removed by reduction. The residual reduced major chain exhibited significant enzymic activity after refolding at low pH had occurred, and most of the native activity was regained upon subsequent oxidation. The catalytic capacity of the chymotrypsins therefore appears to be determined largely by the major chain, the primary structure of which shows extensive homology with that of elastase. Although α-chymotrypsin was found to be irreversibly inactivated by the elastase inhibitor dinitrophenylbromobutanone, a much faster rate of modification of a histidine, rather than a glutamate, residue was observed.     

Ribonuclease structure and catalysis: crystal structure of sulfate-free native ribonuclease A at 1.5-A resolution

The structure of native bovine pancreatic ribonuclease A, without the inhibitory sulfate anion normally bound at the active site, has been determined by X-ray diffraction at 1.53-A resolution. Treatment of a crystal of ribonuclease containing sulfate with an alkaline buffer released most of the sulfate anions. On return to active pH, few of the side chains moved, and the backbone structure remained unchanged. The active site conformation was essentially unchanged except for the replacement of the sulfate anion by a water molecule, which is hydrogen-bonded to histidine-12 and to another water, and for a small movement of the side chain of lysine-41. Histidines-12 and -119, the catalytic basic and acidic residues, have not moved. Thus the distance between them, and the presence of an intervening water, prohibits the possibility of their being hydrogen-bonded together. The structure has been refined by restrained least squares to an R factor of 0.17. Analysis of individual atomic temperature factors indicates that the molecule has become less rigid in general but that some regions were particularly affected by loss of the sulfate, while others were relatively unaffected. The active site geometry of native ribonuclease A supports the original in-line mechanism of Rabin and co-workers and is in disagreement with the adjacent mechanism of Witzel and co-workers.     

Atomic coordinates for tosyl-elastase

The full set of non-hydrogen, atomic coordinates of porcine tosyl elastase is given. This includes the tosyl group and the internal water molecules.     

Structural Similarities between α-Lytic Protease of Myxobacter 495 and Elastase

A model based on the structures of α-chymotrypsin and elastase suggests an explanation for the close resemblance in the catalytic behaviour of these two enzymes.     

Three-dimensional structure of the complex of 4-guanidino-Neu5Ac2en and influenza virus neuraminidase.

The three-dimensional X-ray structure of a complex of the potent neuraminidase inhibitor 4-guanidino-Neu5Ac2en and influenza virus neuraminidase (Subtype N9) has been obtained utilizing diffraction data to 1.8 A resolution. The interactions of the inhibitor, solvent water molecules, and the active site residues have been accurately determined. Six water molecules bound in the native structure have been displaced by the inhibitor, and the active site residues show no significant conformational changes on binding. Sialic acid, the natural substrate, binds in a half-chair conformation that is isosteric to the inhibitor. The conformation of the inhibitor in the active site of the X-ray structure concurs with that obtained by theoretical calculations and validates the structure-based design of the inhibitor. Comparison of known high-resolution structures of neuraminidase subtypes N2, N9, and B shows good structural conservation of the active site protein atoms, but the location of the water molecules in the respective active sites is less conserved. In particular, the environment of the 4-guanidino group of the inhibitor is strongly conserved and is the basis for the antiviral action of the inhibitor across all presently known influenza strains. Differences in the solvent structure in the active site may be related to variation in the affinities of inhibitors to different subtypes of neuraminidase.     

Selective oxidation of Met-192 in bovine α-chymotrypsin. Effect on catalytic and inhibitor binding properties

Catalytic and inhibitor binding properties of bovine α-chymotrypsin, in which the Met-192 residue has been converted by treatment with chloramine T to the sulfoxide derivative (Met(O)192 α-chymotrypsin), have been examined relative to the native enzyme (α-chymotrypsin), between pH 4.5 and 8.0 (μ = 0.1), and/or 5.0°C and 40.0°C. Values of k_cat, k₊₂ and/or k₊₃ for the hydrolysis of all the substrates examined (i.e., tMetAcONp, ZAlaONp, ZLeuONp, ZLysONp and ZTyrONp) catalyzed by native and Met(O)192 α-chymotrypsin are similar, as well as values of K_m for the hydrolysis of ZLeuONp, ZLysONp and ZTyrONp. On the other hand, K_s and K_m values for the hydrolysis of ZAlaONp and tMetAcONp are decreased by about 5-fold. Met-192 oxidation does not affect the kinetic and thermodynamic parameters for the (de)stabilization of the complex formed between the proteinase and the bovine basic pancreatic trypsin inhibitor. On the other hand, the recognition process between between α-chymotrypsin and the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis is influenced by the oxidation event. Considering known molecular models, the observed catalytic and inhibitor binding properties of native and Met(O)192 α-chymotrypsin were related to the inferred stereochemistry of the proteinase-substrate and proteinase-inhibitor contact region(s).     

Solution behavior of α-chymotrypsin dissolved in nonpolar organic solvents via hydrophobic ion pairing

Dissolution of α-chymotrypsin in nonpolar organic solvents can be achieved using hydrophobic ion pairing, whereby the polar counterions are replaced by a stoichiometric number of detergent molecules. Using Aerosol OT[AOT, sodium bis(2-octyl)sulfosuccinate], it is possible to partition significant amounts of the enzyme into alkanes and chlorocarbons. Apparent solubility in isooctane is greater than 1 mg/mL (80 μM). Necessary conditions for achieving effective partitioning of α-chymotrypsin into these solvents are described. Using CD spectroscopy, it can be shown that the AOT–α-chymotrypsin (CMT) complex retains its native secondary and tertiary structure when dissolved in alkanes, and that the globular structure is stable to more than 100°C. In contrast, α-chymotrypsin unfolds at 54°C in aqueous solution. The relative solubility of the AOT–CMT complex in a variety of alkanes and chlorocarbons is also reported. The native structure of α-chymotrypsin is maintained in carbon tetrachloride, but not in methylene chloride or chloroform. © 1995 John Wiley & Sons, Inc.     

Selective oxidation of methionyl residues in the human recombinant secretory leukocyte proteinase inhibitor. Effect on the inhibitor binding properties

Binding of the human recombinant secretory leukocyte proteinase inhibitor (SLPI) [native and with the methionyl residues at positions 73, 82, 94 and 96 of domain 2 oxidized to the sulfoxide derivative (Met(O) SLPI)] to bovine α-chymotrypsin (α-chymotrypsin) [native and with the Met192 residue converted to the sufoxide derivative (Met(O) α-chymotrypsin)] as well as to native bovine β-trypsin (β-trypsin), which does not contain methionyl residues, has been investigated between pH 4.0 and 8.0, and between 10.0°C ad 30.0°C, from thermodynamic and/or kinetic viewpoints. By increasing the number of oxidized methytonyl residues present at the proteinase: inhibitor contact interface (from 0 to 3), the adducts investigated are increasingly destabilized and the relaxation time of the complexes into conformers less stable is enhanced. On the other hand, the selective oxidation methionyl residues of SLPI and α-chymotrypsin, by the reaction with chloramines T, does not affect the proteinase inhibition recognition mechanism. Therefore, even though conformational changes may occur in the conversion native SLPI and native α-chymotrypsin to their Met(O) derivatives, a localized steric hindrance can be considered as the main structural determinant accounting for the reported results.     

Structure of native and apo carbonic anhydrase II and structure of some of its anion-ligand complexes.

In order to obtain a better structural framework for understanding the catalytic mechanism of carbonic anhydrase, a number of inhibitor complexes of the enzyme were investigated crystallographically. The three-dimensional structure of free human carbonic anhydrase II was refined at pH 7.8 (1.54 A resolution) and at pH 6.0 (1.67 A resolution). The structure around the zinc ion was identical at both pH values. The structure of the zinc-free enzyme was virtually identical with that of the native enzyme, apart from a water molecule that had moved 0.9 A to fill the space that would be occupied by the zinc ion. The complexes with the anionic inhibitors bisulfite and formate were also studied at neutral pH. Bisulfite binds with one of its oxygen atoms, presumably protonized, to the zinc ion and replaces the zinc water. Formate, lacking a hydroxyl group, is bound with its oxygen atoms not far away from the position of the non-protonized oxygen atoms of the bisulfite complex, i.e. at hydrogen bond distance from Thr199 N and at a position between the zinc ion and the hydrophobic part of the active site. The result of these and other studies have implications for our view of the catalytic function of the enzyme, since virtually all inhibitors share some features with substrate, product or expected transition states. A reaction scheme where electrophilic activation of carbon dioxide plays an important role in the hydration reaction is presented. In the reverse direction, the protonized oxygen of the bicarbonate is forced upon the zinc ion, thereby facilitating cleavage of the carbon-oxygen bond. This is achieved by the combined action of the anionic binding site, which binds carboxyl groups, the side-chain of threonine 199, which discriminates between hydrogen bond donors and acceptors, and hydrophobic interaction between substrate and the active site cavity. The required proton transfer between the zinc water and His64 can take place through water molecules 292 and 318.     

Effect of anesthetics on the stability of oxyhemoglobin S

The conversion of the serine-195 in α-chymotrypsin to dehydroalanine results in two conformational substates that differ in their extinction coefficients at 240nm. The active site methionine-192 in the substate with lower absorption at 240nm is alkylated by α-bromo-4-nitroacetophenone at a rate of 7.0×10^?4sec^?1, similar to that found for α-chymotrypsin; the substate with higher absorption at 240nm reacts 14 times slower. These two substates are not separated by an affinity resin containing lima bean trypsin inhibitor. These data infer that the serine-195 plays a role in the stabilization of the active site conformation in α-chymotrypsin.     

Consequences of the modification of tryptophan-215 on the function of bovine alpha-chymotrypsin

At pH values between 4.5 and 7.0, 2-hydroxy-5-nitrobenzyl bromide reacts selectively with tryptophan-215 in bovine α-chymotrypsin as demonstrated by the isolation of peptides containing modified amino acid residues. The degree of substitution at lower pH values indicates conformational changes in the enzyme observed previously by physico-chemical methods. The substitution of the native enzyme results in the loss of esterase activity. Nevertheless 2-hydroxy-5-nitro-benzyl chymotrypsin is still able to react with diisopropylphosphofluoridate.The catalytically inactive derivatives of α-chymotrypsin, DIP, TPCK and anhydro-chymotrypsin, as well as the complex of α-chymotrypsin with basic pancreatic trypsin inhibitor, are not modified by 2-hydroxy-5-nitrobenzyl bromide under the same conditions as those used for the native enzyme.HNB-chymotrypsin and anhydro-chymotrypsin, both catalytically inactive, form stoichiometric complexes with the basic pancreatic trypsin inhibitor whereas both PMS and DIP α-chymotrypsin did not have this complexing property. From the results of this and a preceding study (Ako et al., 1972) it is concluded that the intactness of the catalytic function of ehymotrypsin is not obligatory for the binding of basic pancreatic inhibitor.     

Three-dimensional structure of the elastase of Pseudomonas aeruginosa at 1.5-A resolution

Pseudomonas aeruginosa elastase (PAE) is a zinc metalloprotease with 301 amino acids. We have crystallized and solved the three-dimensional structure of PAE, using data to 1.5-A resolution, and have refined the native molecular structure to R = 0.188. The overall tertiary structure of the PAE molecule is similar to that of thermolysin, with which it shares 28% amino acid sequence identity. Nearly all of the active site residues that might potentially interact with substrates are identical in the two proteins. However, the active site cleft is significantly more "open" in PAE than in thermolysin.     

A Bowman-Birk inhibitor with anti-elastase activity from Lathyrus sativus L. seeds

Four Bowman-Birk inhibitors, named LSI-1/4, were isolated and purified from Lathyrus sativus L. seeds. The purification procedure consisted of two cation-exchange chromatography steps, followed by gel-filtration and RP-HPLC. Mass spectrometry analysis of LSI-1/4 inhibitors yielded relative molecular masses of 7914.41 for LSI-1, 6867.67 for LSI-2, 7341.24 for LSI-3 and 7460.01 for LSI-4. N-terminal sequences (up to 30 residues) of LSI-1/4 inhibitors were identical with the exception of sequence positions 21, 27 and 28 and highly similar to those of other Bowman-Birk inhibitors isolated from Leguminosae plants. Inhibitors LSI-1/4 were active towards trypsin and α-chymotrypsin, with IC(50) values for 12.6 nM of trypsin ranging from 4.9 to 24.3 nM. A lower activity was observed against bovine α-chymotrypsin (IC(50) values ranging from 0.5 to 3.4 μM for 15.0 nM of α-chymotrypsin). Peptide mapping of the LSI-1 sequence showed the presence of an Ala residue in the second reactive site, thus explaining the low anti-chymotrypsin activity of this inhibitor. In addition, LSI-1 was endowed with anti-elastase activity, being able to inhibit human leukocyte elastase.     

The three-dimensional structure of the native ternary complex of bovine pancreatic procarboxypeptidase A with proproteinase E and chymotrypsinogen C.

The metalloexozymogen procarboxypeptidase A is mainly secreted in ruminants as a ternary complex with zymogens of two serine endoproteinases, chymotrypsinogen C and proproteinase E. The bovine complex has been crystallized, and its molecular structure analysed and refined at 2.6 A resolution to an R factor of 0.198. In this heterotrimer, the activation segment of procarboxypeptidase A essentially clamps the other two subunits, which shield the activation sites of the former from tryptic attack. In contrast, the propeptides of both serine proproteinases are freely accessible to trypsin. This arrangement explains the sequential and delayed activation of the constituent zymogens. Procarboxypeptidase A is virtually identical to the homologous monomeric porcine form. Chymotrypsinogen C displays structural features characteristic for chymotrypsins as well as elastases, except for its activation domain; similar to bovine chymotrypsinogen A, its binding site is not properly formed, while its surface located activation segment is disordered. The proproteinase E structure is fully ordered and strikingly similar to active porcine elastase; its specificity pocket is occluded, while the activation segment is fixed to the molecular surface. This first structure of a native zymogen from the proteinase E/elastase family does not fundamentally differ from the serine proproteinases known so far.     

Refined structure of dienelactone hydrolase at 1.8 A

The structure of dienelactone hydrolase (DLH) from Pseudomonus sp. B13, after stereochemically restrained least-squares refinement at 1.8 A resolution, is described. The final molecular model of DLH has a conventional R value of 0.150 and includes all but the carboxyl-terminal three residues that are crystallographically disordered. The positions of 279 water molecules are included in the final model. The root-mean-square deviation from ideal bond distances for the model is 0.014 A and the error in atomic co-ordinates is estimated to be 0.15 A. DLH is a monomeric enzyme containing 236 amino acid residues and is a member of the beta-ketoadipate pathway found in bacteria and fungi. DLH is an alpha/beta protein containing seven helices and eight strands of beta-pleated sheet. A single 4-turn 3(10)-helix is seen. The active-site Cys123 residues at the N-terminal end of an alpha-helix that is peculiar in its consisting entirely of hydrophobic residues (except for a C-terminal lysine). The beta-sheet is composed of parallel strands except for strand 2, which gives rise to a short antiparallel region at the N-terminal end of the central beta-sheet. The active-site cysteine residue is part of a triad of residues consisting of Cys123, His202 and Asp171, and is reminiscent of the serine/cysteine proteases. As in papain and actinidin, the active thiol is partially oxidized during X-ray data collection. The positions of both the reduced and the oxidized sulphur are described. The active site geometry suggests that a change in the conformation of the native thiol occurs upon diffusion of substrate into the active site cleft of DLH. This enables nucleophilic attack by the gamma-sulphur to occur on the cyclic ester substrate through a ring-opening reaction.     

High yield chromatographic purification of enzymatically produced tyrosine fructosyl ester

The synthesis of L-tyrosine fructosyl ester, from fructose and L-tyrosine methyl ester, was carried out by a transesterification reaction catalyzed by α-chymotrypsin in water without any organic cosolvent. The yield was optimized by regulating the pH?of the reaction medium and a maximum yield of 63% was obtained. A two-step process of tyrosine fructosyl ester purification was proposed using adsorbent resin and activated carbon. Different elutions and supports easily applicable to industrial process have been studied. Solutions of tyrosine fructosyl ester with a 96% purity and 70% recovery yield were obtained by chromatography on a column of acrylic polyester resins with a solution of 0.5?M NaCl as eluant.     

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.     

Simulations of dynamical properties of a Michaelis complex: porcine pancreatic elastase and the hexapeptide, Thr-Pro-n Val-Leu-Tyr-Thr

Molecular dynamic simulations (30ps) of the Michaelis complex of hexapeptide (Thr-Pro-nVal-Leu-Tyr-Thr) bound to porcine pancreatic elastase (PPE) hydrated by about 2000 water molecules have been performed using the AMBER 3.0 program package. Dynamical properties of the conformation of the active site have been examined. A comparison with previously reported simulations of native PPE shows that after the substrate is bound, the catalytically crucial H-bond between O gamma-H group of (Ser 195) and nitrogen N epsilon (His 57) is more readily formed. These results show, however, that the H-bond does not adopt the most favorable conformation. The O gamma-H group of Ser 195 has a statistical preference for an attractive interaction with the O = C carbonyl (Ser 214) rather than the nitrogen N epsilon (His 57).     

Refined crystal structure of γ-chymotrypsin at 1.9 Å resolution: Comparison with other pancreatic serine proteases

The crystal structure of γ-chymotrypsin, the monomeric form of chymotrypsin, has been determined and refined to a crystallographic R-factor of 0.18 at 1.9 Å resolution. The details of the catalytic triad involving Asp102, His57 and Ser195 agree well with the results found for trypsin (Chambers & Stroud, 1979) and Streptomyces griseus protease A (Sielecki et al., 1979). As in many of the other serine proteases, the Oγ of Ser195 does not appear to be hydrogen-bonded to His57.The three-dimensional structures of γ- and α-chymotrypsin (Birktoft & Blow, 1972) are closely similar. The largest backbone differences occur in the “calcium binding loop” (residues 75 to 78) and in the “autolysis loop” (residues 146, 149 and 150). Ala149 and Asn150 are disordered in γ-chymotrypsin, whereas they are stabilized by intermolecular interactions in α-chymotrypsin. The conformation of Ser218 is also different, presumably the indirect result of the dimeric interactions of α-chymotrypsin. These results are discussed in terms of the slow, pH-dependent interconversion of α- and γ-chymotrypsin.