Prediction of protein--ligand interactions: the complex of porcine pancreatic elastase with a valine-derived benzoxazinone

Prior to availability of the crystal structure of the complex, we evaluated models of the complex between porcine pancreatic elastase and a t-Boc–Val-derived benzoxazinone inhibitor. Models of the noncovalent and covalent complex were generated using computer graphics and each model was subjected to energy minimization using molecular mechanics. After the crystal structure became available, we found that the model with the lowest energy was in good agreement with the crystal structure, except for the position of the His57 side chain. Permissible conformations of the inhibitor were based on information from x-ray crystal structures and an earlier conformational energy investigation of t-Boc–amino acids. We did not, however, limit ourselves to these conformations. The conformation of the inhibitor in the lowest energy model and crystal structure, was not similar to any of the minimum-energy conformations of t-Boc–amino acids. This suggests that limiting proposed binding modes only to the lowest energy conformations of a ligand (prior to binding) may sometimes unfairly bias the procedure.     

The folding of pancreatic elastase: Independent domain refolding and inter-domain interaction

The role of domains in the refolding of elastase, a two domain protein, was investigated. It has been demonstrated that fragment 126–245, corresponding to one of the two domains, is able to refold independently. Moreover, the in vitro complementation of the two domains lead to a molecule having the overall conformation of the native protein and only a weak but significant activity.     

Evidence for the amino acid sequence of porcine pancreatic elastase

The preparation and purification of tryptic peptides from aminoethylated Dip-elastase and [(14)C]carboxymethylated Dip-elastase, and of peptic peptides from native elastase is described. A summary of the results of chemical studies used to elucidate the amino acid sequence of these peptides is presented. Full details are given in a supplementary paper that has been deposited as Supplementary Publication SUP 50016 at the National Lending Library for Science and Technology, Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 1-20. These results, together with those from previously published papers, are used to establish the complete amino acid sequence of elastase, which is a single polypeptide chain of 240 residues, molecular weight 25900, containing four disulphide bridges.     

The effects of various cations on the survival of brown trout, Salmo trutta at low pHs

The survival times of hatchery reared year-old brown trout ( Salmo trutta ) were tested in solutions containing various ions at pHs between 3.5 and 4.0. The effect of calcium in prolonging the survival time compared with that in deionized water at all pHs tested, was more marked than that of sodium which was only effective at pH 4.0. Potassium and magnesium had no effect individually, but aluminium prolonged the survival time in sodium solutions. Fish from a naturally acid river (Tovdal-Norway) survived significantly longer than hatchery reared fish, indicating the importance of the previous history of the fish to their sensitivity to low pH.     

Structure of the product complex of acetyl-Ala-Pro-Ala with porcine pancreatic elastase at 1.65 A resolution

A single crystal of porcine pancreatic elastase was mounted in a thin-walled capillary and allowed to react with acetyl-Ala-Pro-Ala-paranitroanalide. Diffraction data to 1.65 A resolution were measured and the isomorphous structure was solved from the difference Fourier map. The structure contains two surprises. Two molecules of the product: acetyl-Ala-Pro-Ala molecule are bound in the extended binding site. Both molecules are bound backwards with respect to the established mode of peptide binding.     

Catalytic properties of porcine pancreatic elastase: a steady-state and pre-steady-state study

Pre-steady-state and steady-state kinetics for the p.p. elastase-catalysed hydrolysis of ZAlaONp, one of the most favourable substrates for this serine protease, have been studied between pH 4.0 and 8.0. The results are consistent with the minimum three-step mechanism: (formula; see text) Under pre-steady-state conditions, where [E0] much greater than [S0], the values of the dissociation constant of the E X S complex (Ks = k-1/k+1) and of the individual rate constants for the catalytic steps (k+2 and k+3) have been determined over the whole pH range explored. Under steady-state conditions, where [S0] much greater than [E0], the values of kcat and Km have been obtained over the same pH range. The pH profiles of k+2, k+3, k+2/Ks, kcat, kcat/Km reflect the ionization of a group, probably His57, with a pKa value of 6.85 +/- 0.10. The values of Ks and Km are pH independent. The steady-state parameters for the p.p. elastase-catalysed hydrolysis of a number of p-nitrophenyl esters of N-alpha-carbobenzoxy-L-amino acids have been also determined between pH 4.0 and 8.0 and compared with those of b.beta-trypsin and b.alpha-chymotrypsin. For all the substrates examined the acylation step (k+2) is rate limiting in the p.p. elastase catalysis, between pH 4.0 and 8.0. The different catalytic behaviours of p.p. elastase, b.beta-trypsin and b.alpha-chymotrypsin are consistent with the known three-dimensional structures of these serine proteases.     

Role of the environment in the refolding of reduced pancreatic trypsin inhibitor

Refolding of reduced pancreatic trypsin inhibitor has been examined under a variety of environmental conditions, varying the temperature, pH and ions of the solution, and determining the transient intermediates that accumulate and the kinetics of refolding. The effects of these variables on the rate of the thiol-disulphide exchange reaction, which is involved in each refolding step observed, were determined so that the kinetic effects on refolding could be interpreted in terms of the effect on protein conformation.Low temperature favoured the initial one-and two-disulphide intermediates with native-like disulphide bonds; the differences in enthalpy, entropy and heat capacity of the various species were estimated. Varying the pH somewhat had little effect on the pathway, as did variation of the ionic strength, although there were significant effects on the reactivities of various cysteine thiol groups at low ionic strength, which were apparently due to enhanced electrostatic interactions between charged groups of the protein. Varying the ions of the solution according to the Hofmeister series produced effects like those observed by others on protein stability: stabilizing salts produced the same effect as lower temperatures, destabilizing salts as higher temperatures, while indifferent salts had little effect. Low concentrations of the denaturants urea and guanidinium chloride had effects similar to those of destabilizing Hofmeister salts.All these effects point to the important intermediate states that are most populated having the greatest extent of stabilizing hydrophobic interactions.     

Hydrogen kinetics of peptide amide protons at the bovine pancreatic trypsin inhibitor protein-solvent interface

Hydrogen exchange rate constants of the 25 most rapidly exchanging peptide amide protons in bovine pancreatic trypsin inhibitor have been determined over a range of pH that spans pH min, the pH of minimum rate. Most of these are on the protein surface, exposed to solvent and not hydrogen bonded in the crystal structure. Contrary to commonly held assumptions, the exchange kinetics of surface NH groups are not equivalent to the kinetics of NH groups in peptides in the extended configuration. All surface NH groups exchange more slowly than NH groups in model peptides, with rate constants distributed over a range of more than two orders of magnitude. In addition, their pH min values vary widely. For most of the surface NH groups, pH min is lower than in model compounds and, for several, pH min is less than 1. These results indicate that the local environment of the surface peptide groups when the exchange event occurs is very different from that of extended peptides. Analysis based on consideration of an O-protonation mechanism for acid catalysis and of electrostatic effects on exchange kinetics further indicates (see the accompanying paper) that, in general, exchange of surface NH groups occurs from a conformation of the protein approximated by the crystal structure. The 1H-2H exchange rate constants were measured from 300 MHz nuclear magnetic resonance spectra in which assigned surface N1H resonances are resolved by the use of partially deuterated protein samples. A marked pH dependence of the chemical shifts observed in the pH range 1 to 4.5 for several surface NH groups reflects the titration of nearby carboxyl groups.     

The atomic structure of crystalline porcine pancreatic elastase at 2.5 A resolution: comparisons with the structure of alpha-chymotrypsin

Three isomorphous heavy-atom derivatives have been used to calculate a 2.5 Å resolution electron density map of tosyl-elastase at pH 5.0, from which an accurate atomic model has been constructed. Atomic co-ordinates measured from this model have been refined using model building, real-space refinement and energy minimization programs. The three-dimensional conformation of the polypeptide chain is described in terms of conformational angles, hydrogen-bonding networks and the environment of different types of amino acid side-chain.Difference Fourier calculation of the high resolution structure of native elastase at pH 5.0 shows it to be virtually identical to that of the tosyl derivative, except near the tosyl group. The conformation of the catalytically important residues in native elastase is very similar to that of native α-chymotrypsin, except for the orientation of the active centre serine oxygen. The significance of important structural similarities and differences between these two enzymes is discussed.Elastase contains 25 internal water molecules which play an important role in stabilizing the active conformation of the enzyme. Many of these water molecules are in identical positions to those found in the interior of α-chymotrypsin     

The primary structure of porcine pancreatic elastase: The N-terminus and disulphide bridges

1. The amino acid composition and N-terminal groups of purified elastase show that it is a single peptide chain of 234 residues. 2. The N-terminal sequence is Val-Val-Gly-Gly-Thr-Glu-. 3. The sequences around the four disulphide bridges were determined by using a ;diagonal' electrophoretic technique. 4. These four bridges are homologous with the four common to bovine trypsin and chymotrypsin. 5. Out of 83 residues of the elastase sequence so far determined, 43 are homologous with similar regions of trypsin and chymotrypsin. 6. The evolutionary ancestry of these enzymes is discussed.     

Specificity of elastases: degradation of the oxidized beta-chain of insulin by porcine pancreatic elastase II and dog leucocyte elastase.

Porcine elastase II (EC 3.4.21.-), a pancreatic proteinase with elastolytic activity, hydrolyses the oxidized beta-chain of insulin with major cleavages occurring at Leu17-Val18, Phe24-Phe25, Phe25-Tyr26 and Tyr26-Thr27. Canine leucocytic elastase splits the same substrate with major sites at Val12-Glu13 and Val18-Cys19 O3H. This indicates similarity of elastase II to chymotrypsins (EC 3.4.21.1 or 3.4.21.2) and of dog leucocyte enzyme to human granulocyte elastase and porcine pancreatic elastase I (EC 3.4.21.11).     

The crystal structure of the complex of non-peptidic inhibitor of human neutrophil elastase ONO-6818 and porcine pancreatic elastase

The crystal structure of a new inhibitor of human neutrophil elastase (HNE), N-[2-[5-(tert-butyl)-1,3,4-oxadiazol-2-yl]-(IRS)-1-(methylethyl)-2-oxoethyl]-2-(5-amino-6-oxo-2-phenyl-6H-pyrimidin-1-ly)acetamide (ONO-6818, 1) complexed to porcine pancreatic elastase (PPE) has been determined at 1.86 A resolution. Analytical results provided evidence of a 1:1 complex in which the electrophilic ketone of 1 covalently bound to O gamma of Ser195 at the active site of PPE. The role of the unique electron-withdrawing ketone of 1 has been elucidated.     

X-ray diffraction analysis of the inhibition of porcine pancreatic elastase by a peptidyl trifluoromethylketone

X-ray crystallographic data to 2.57 A resolution (1 A = 0.1 nm) have been measured for the complex of a peptidyl trifluoromethylketone inhibitor with porcine pancreatic elastase (PPE); R = 0.14. The inhibitor forms a stable complex with the enzyme by means of a covalent attachment to active site Ser195O gamma, resulting in a hemiketal moiety with tetrahedral geometry. The tripeptide protion binds as an antiparallel beta-sheet, with four hydrogen bonds augmenting the active-site covalent linkage, Ki = 9.5 microM. His57 exhibits a bifurcated H-bond to both Ser195O gamma and an F atom of the inhibitor. This study is one of a series which explores the binding geometry of a variety of small substrates and inhibitors to PPE. This peptidyl-PPE complex affords insight into the binding geometry of a novel trifluoromethylketone moiety to a serine proteinase.     

Crystal structures of the complex of porcine pancreatic elastase with two valine-derived benzoxazinone inhibitors

The crystal structures of porcine pancreatic elastase complexed to two similar benzoxazinone inhibitors are reported to 2.09 A and 1.76 A resolution, and refined to conventional R factors of 0.153 and 0.172.     

Nucleophilic cleavage of the complex between human alpha-1-antitrypsin and porcine pancreatic elastase

Several nucleophilic amines were examined to determine their ability to split the alpha-1-antitrypsin-elastase complex. Hydrazine and hydroxylamine, their methyl derivatives, and methylamine were tested in the pH range of 8 to 10.6. Only hydrazine and methylamine produced complete and clean cleavage of the complex into its inhibitor and enzyme components. When [¹⁴C]-methylamine at pH 10.6 was used about 0.3 mol of the nucleophile was specifically bound per mol of the inhibitor component. An interfering reaction between methylamine and the native inhibitor was controlled by preincubation with unlabeled methylamine.     

The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions.

Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure. The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank. The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed. It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area. The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure. An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms. The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions.     

Isolation and expression in Escherichia coli of a cDNA clone encoding porcine pancreatic elastase

We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum.     

Pulmonary damage in the rabbit caused by porcine pancreatic elastase and leukocyte lysate: unusual microscopic aspects]

Intratracheal injection of rabbit whole leukocyte homogenate induced emphysema-like lesions in rabbits. The lesions were produced only by preparations having elastinolytic activity. The pathological aspects appeared similar to those induced by the administration of porcine pancreatic elastase. The pulmonary changes, resembling several of the anatomic appearances of panacinar human emphysema, may be a suitable experimental model for studying histogenesis of panacinar human emphysema. Numerous abnormal fenestrations were present in air spaces walls and were a constant feature in opposition to that has been reported in elastase-treated hamsters. For the presence of a prominent dilatation of the periarterial lymphatic network, this experimental model might be used also for studying the ultrastructural features of pulmonary lymphatic vessels.