The role of peripheral interactions in chymosin specificity

Kinetic parameters for the splitting of model peptide substrates and chi-casein with chymosin have been interpreted on the basis of the three-dimensional structure of chymosin. Model peptide substrates contain a fragment of the chi-casein sequence in the region of the bond Phe-105--Met-106 splitted with the enzyme. It was shown that the possible reason of the enormous milk-clotting efficiency of chymosin may be partly associated with the electrostatic interaction of the positive charged segment 98-102 (His-Pro-His-Pro-His) of the substrate and outer loop of the enzyme which contains Glu-245, Asp-247, Asp-249, Asp-251.     

Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica

Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa. It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. K m arg and Vmax values for the recombinant two-domain AK were determined. These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193. Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli. Domain 2 still exhibited activity. However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK. Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly. Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence. Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures. We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values. The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax. These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.     

Crystal structures of Aspergillus oryzae aspartic proteinase and its complex with an inhibitor pepstatin at 1.9A resolution

The X-ray structures of Aspergillus oryzae aspartic proteinase (AOAP) and its complex with inhibitor pepstatin have been determined at 1.9A resolution. AOAP was crystallized in an orthorhombic system with the space group P2(1)2(1)2(1) and cell dimensions of a=49.4A, b=79.4A, and c=93.6A. By the soaking of pepstatin, crystals are transformed into a monoclinic system with the space group C2 and cell dimensions of a=106.8A, b=38.6A, c=78.7A, and beta=120.3 degrees. The structures of AOAP and AOAP/pepstatin complex were refined to an R-factor of 0.177 (R(free)=0.213) and of 0.185 (0.221), respectively. AOAP has a crescent-shaped structure with two lobes (N-lobe and C-lobe) and the deep active site cleft is constructed between them. At the center of the active site cleft, two Asp residues (Asp33 and Asp214) form the active dyad with a hydrogen bonding solvent molecule between them. Pepstatin binds to the active site cleft via hydrogen bonds and hydrophobic interactions with the enzyme. The structures of AOAP and AOAP/pepstatin complex including interactions between the enzyme and pepstatin are very similar to those of other structure-solved aspartic proteinases and their complexes with pepstatin. Generally, aspartic proteinases cleave a peptide bond between hydrophobic amino acid residues, but AOAP can also recognize the Lys/Arg residue as well as hydrophobic amino acid residues, leading to the activation of trypsinogen and chymotrypsinogen. The X-ray structure of AOAP/pepstatin complex and preliminary modeling show two possible sites of recognition for the positively charged groups of Lys/Arg residues around the active site of AOAP.     

Optimization of the catalytic properties of Aspergillus fumigatus phytase based on the three-dimensional structure

Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x [mg protein](-1) at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.     

Protein engineering of chymosin; modification of the optimum pH of enzyme catalysis

The aspartic proteinase chymosin exhibits a local network of hydrogen bonds involving the active site aspartates and surrounding residues which may have an influence on the rate and optimal pH of substrate cleavage. We have introduced into chymosin B the following substitutions: Asp304 to Ala (D304A), Thr218 to Ala (T218A) and Gly244 to Asp (G244D, chymosin A), using oligonucleotide-directed mutagenesis. Kinetic analysis of these active mutants shows shifts in their pH optima to 4.4 D304A, 4.2 T218A and 4.0 G244D compared with 3.8 for chymosin B using a synthetic octapeptide substrate. The upward shift of the D304A and T218A may be due to the loss of hydrogen bond interactions indirectly affecting the catalytic aspartates 32 and 215. The G244D mutation which is in a flexible loop on the surface of the enzyme may alter the conformation of the specificity pockets on the prime side of the scissile bond.     

Probing the function of conserved residues in the serine/threonine phosphatase PP2Calpha

Human PP2Calpha is a metal-dependent phosphoserine/phosphothreonine protein phosphatase and is the representative member of the large PPM family. The X-ray structure of human PP2Calpha has revealed an active site containing a dinuclear metal ion center that is coordinated by several invariant carboxylate residues. However, direct evidence for the catalytic function of these and other active-site residues has not been established. Using site-directed mutagenesis and enzyme kinetic analyses, we probed the roles of conserved active-site amino acids within PP2Calpha. Asp-60 bridges metals M1 and M2, and Asp-239 coordinates metal M2, both of which were replaced individually to asparagine residues. These point mutations resulted in >or=1000-fold decrease in k(cat) and >or=30-fold increase in K(m) value for Mn(2+). Mutation of Asp-282 to asparagine caused a 100-fold decrease in k(cat), but no significant effect on K(m) values for metal and substrate, consistent with Asp-282 activating a metal-bound water nucleophile. Mutants T128A, E37Q, D38N, and H40A displayed little or no alterations on k(cat) and K(m) values for substrate or metal ion (Mn(2+)). Analysis of H62Q and R33A yielded k(cat) values that were 20- and 2-fold lower than wild-type, respectively. The mutant R33A showed a 8-fold higher K(m) for substrate, while the K(m) observed with H62Q was unaffected. A pH-rate profile of the H62Q mutant showed loss of the ionization that must be protonated for activity. Br?nsted analysis of substrate leaving group pK(a) values for H62Q indicated a greater dependency (slope -0.84) on leaving group pK(a) in comparison to wild-type (slope -0.33). These data provide strong evidence that His-62 acts as a general acid during the cleavage of the P-O bond.     

Peptide substrates for chymosin (rennin). Interaction sites in kappa-casein-related sequences located outside the (103-108)-hexapeptide region that fits into the enzyme''s active-site cleft.

The role of individual amino acid residues in the 98-102 and 111-112 regions of bovine kappa-casein in its interaction with the milk-clotting enzyme chymosin (rennin) was investigated. to this end the tryptic 98-112 fragment of kappa-casein was modified in its N- and/or C-terminal part by chemical (guanidation, ethoxyformylation, repeated Edman degradation) and enzymic (carboxypeptidase) treatments. Further, use was made of short synthetic kappa-casein analogues in which His-102 had been replaced by Pro or Lys. All peptides and their derivatives were tested comparatively at various pH values for their ability to act as chymosin substrates via specific cleavage of the peptide bond at position 105-106. The results indicate that in the alternating 98-102 sequence (His-Pro-His-Pro-His) the His as well as the Pro residues contribute to the substrate activity with no predominant role of any one of these groups. Another interaction site is formed by the Lys residue at position 111 of the substrate. A model of the enzyme-substrate complex is proposed. Herein the 103-108 fragment of the substrate, to be accommodated within the enzyme's active-site cleft, is brought into position by electrostatic binding (via His-98, His-100, His-102 and Lys-111) near the entrance of the cleft. These interactions are strongly supported by Pro residues at positions 99, 101, 109 and 110 of the substrate, which act as stabilizers of the proper conformation of the substrate in the enzyme-substrate complex.     

Evolution in the structure and function of carboxyl proteases

Summary A model for the structure and function of extracellular carboxyl (acid) proteases can be established from three amino acid sequences and four crystal structures of these enzymes. The carboxyl proteases from gastric and fungal origins are very homologous in both primary and tertiary structures. The molecules consist of about 320 residues organized with a secondary structure which is primarily comprised of -strands and very similar tertiary structures. An apparent binding cleft, which can accommodate a substrate with about eight amino acid residues, contains near its midpoint the active center residues Asp-215, Asp-32, and Ser-35. These three residues are hydrogen bonded to each other.An intracellular carboxyl protease, cathepsin D, is very homologous to the extracellular enzymes in N-terminal amino acid sequence and primary structure location of active center residues. The tertiary structure of cathepsin D is probably similar, as well. However, cathepsin D contains a unique hydrophobic tail made up of about 100 residues added on the C-terminal side. Cathepsin D precursor is over 100,000 daltons in molecular weights, as contrasted to the gastric carboxyl protease zymogens, which are about 40,000 daltons.     

Human lysosomal alpha-glucosidase. Characterization of the catalytic site.

The substrate analogue conduritol B epoxide (CBE) is demonstrated to be an active site-directed inhibitor of human lysosomal alpha-glucosidase. A competitive mode of inhibition is obtained with glycogen as natural and 4-methylumbelliferyl-alpha-D-glucopyranoside as artificial substrate. The inactivation of the enzyme is time and concentration dependent and results in the covalent binding of CBE. Catalytic activity is required for binding to occur. CBE-labeled peptides containing the catalytic residue of lysosomal alpha-glucosidase were isolated and identified by microsequencing and amino acid analysis. The peptides appeared to originate from a protein domain which is highly conserved among alpha-amylases, maltase, glucoamylases, and transglucanosylases. Based on the sequence similarity and the mechanism of CBE binding, Asp-518 is predicted to be the essential carboxylate in the active site of lysosomal alpha-glucosidase. The functional importance of Asp-518 and other residues around the catalytic site was studied by expression of in vitro mutagenized alpha-glucosidase cDNA in transiently transfected COS cells. Substitution of Asp-513 by Glu-513 is shown to interfere with the posttranslational modification and the intracellular transport of the alpha-glucosidase precursor. The residues Trp-516 and Asp-518 are demonstrated to be critical for catalytic function.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).     

Analysis of the active center of branching enzyme II from maize endosperm

Analysis of the primary structure of mBEII, with those of other branching and amylolytic enzymes as reference, identifies four highly conserved regions which may be involved in substrate binding and in catalysis. When one of the amino acid residues corresponding to the putative catalytic sites of mBEII, i.e., Asp-386, Glu-441, and Asp-509, was replaced, activity disappeared. These putative catalytic residues are located in three different regions (regions 2–4) of the four highly conserved regions (regions 1–4) which exist in the primary structure of most starch hydrolases and related enzymes, including branching enzymes. Region 3, which contains Glu-441 as one of the putative catalytic residues, was located downstream of the carboxyl-terminal position previously reported. The importance of the carboxyl amino acid residues was also demonstrated by chemical modification of the branching enzyme protein using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.     

Investigations on the activation of bovine prochymosin.

Activation of prochymosin at pH below 2.5 results in formation of the active enzyme pseudochymosin by proteolytic cleavage of the bond 27--28. Pseudochymosin is 15 amino acid residues longer than chymosin. It is the final activation product at low pH, whereas chymosin is formed by activation between pH 4 and 5. Pseudochymosin is converted to chymosin when it is brought to pH 5.5. Our present knowledge does not allow quantitative evaluation of the possible reactions involved in formation of pseudochymosin, but the course of activation at pH 2 is in accordance with an intermolecular reaction between two zymogen molecules as the predominant reaction. We find indications of an intramolecular reaction when intermolecular reactions are prevented by immobilization of the zymogen.     

The refined structure of the complex between adenylate kinase from beef heart mitochondrial matrix and its substrate AMP at 1.85 A resolution

The crystal structure of the complex between adenylate kinase from bovine mitochondrial matrix and its substrate AMP has been refined at 1.85 A resolution (1 A = 0.1 nm). Based on 42,519 independent reflections of better than 10 A resolution, a final R-factor of 18.9% was obtained with a model obeying standard geometry within 0.016 A in bond lengths and 3.2 degrees in bond angles. There are two enzyme: substrate complexes in the asymmetric unit, each consisting of 226 amino acid residues, one AMP and one sulfate ion. A superposition of the two full-length polypeptides revealed deviations that can be described as small relative movements of three domains. Best superpositions of individual domains yielded a residual overall root-mean-square deviation of 0.3 A for the backbone atoms and 0.5 A for the sidechains. The final model contains 381 solvent molecules in the asymmetric unit, 2 x 72 = 144 of which occupy corresponding positions in both complexes.     

Purification, characterization, and nucleotide sequence of the thermolabile alpha-amylase from the antarctic psychrotroph Alteromonas haloplanctis A23.

The alpha-amylase excreted by the antarctic bacterium Alteromonas haloplanctis was purified and the corresponding amy gene was cloned and sequenced. N- and C-terminal amino acid sequencing were used to establish the primary structure of the mature A. haloplanctis alpha-amylase which is composed of 453 amino acids with a predicted Mr of 49,340 and a pI of 5.5. Three Ca2+ ions are bound per molecule and its activity is modulated by chloride ions. Within the four consensus sequences, Asp-174, Glu-200, and Asp-264 are the proposed catalytic residues. The psychrotrophic A. haloplanctis alpha-amylase is characterized by a high amylolytic activity at low temperatures, a reduced apparent optimal temperature, and typical thermodynamic activation parameters A. haloplanctis alpha-amylase has also a low thermal stability as demonstrated by the temperature effect on both activity and secondary structure. It is suggested that structure flexibility and lower sensitivity of secondary structure to temperature variations in the low temperature range are the main structural adaptations of the psychrotrophic enzyme. The unusual stacking of small amino acids around the catalytic residues is proposed as a factor inducing active site flexibility and concomitant high activity of the enzyme at low temperatures.     

Effect of point substitutions of Asp-714 and Asp-720 residues on the structure and function of the H+-ATPase of the yeast plasma membrane

Membrane-spanning M5 and M6 segments, which play a role in the formation of cation transport sites in H⁺-, Ca²⁺-, K⁺-, Na⁺-, and other P2-ATPases, are connected by a short extracytoplasmic loop. In the yeast plasma membrane H⁺-ATPase, which belongs to a family of P2-ATPases, the loop is connected to M5 and M6 through the Asp-714 and Asp-720 residues. In this work, the effect of point amino acid replacements of Asp-714 and Asp-720 by Ala, Val, Asn, and Glu residues on the function of the enzyme was studied. The D714A point mutant possessed activities similar to those of the wild-type enzyme, whereas the replacement of Asp-714 by other amino acid residues disrupted biogenesis and led to a loss of activity. All mutants with substitution of Asp-720 were expressed and possessed relatively high activity. The D720V mutant displayed significantly reduced expression level, activity, H⁺ transport and its coupling to ATP hydrolysis. Thus, substitutions of Asp-714, except for the D714N mutant, led to significant defects in biogenesis and/or function of the enzyme. The results indicate the important role for the Asp-714 residue in biogenesis, structure stability, and enzyme function.     

Characterization and study of a κ-casein-like chymosin-sensitive linkage

The present report is dealing with the identification, in various unrelated proteins, of protein fragments sharing local sequence and structure similarities with the chymosin-sensitive linkage surrounding the Phe-Met/Ile bond of κ-caseins. In all these proteins, this linkage is observed within an exposed β-strand-like structure, as also predicted for κ-caseins. The structure of one of these fragments, included in glutamine synthetase, particularly superimposes well with the conformation observed for a chymosin inhibitor (CP-113972) within the complex it forms with chymosin and can be similarly accommodated by specificity pockets within the enzyme substrate binding cleft. The effect of the enzyme activity of chymosin was thus tested on glutamine synthetase. Chymosin cut the latter at the Phe-Met linkage, suggesting that this system may locally resemble the κ-casein/chymosin complex.     

Phenylalanine dehydrogenase from Rhodococcus sp. M4: high-resolution X-ray analyses of inhibitory ternary complexes reveal key features in the oxidative deamination mechanism

The molecular structures of recombinant L-phenylalanine dehydrogenase from Rhodococcus sp. M4 in two different inhibitory ternary complexes have been determined by X-ray crystallographic analyses to high resolution. Both structures show that L-phenylalanine dehydrogenase is a homodimeric enzyme with each monomer composed of distinct globular N- and C-terminal domains separated by a deep cleft containing the active site. The N-terminal domain binds the amino acid substrate and contributes to the interactions at the subunit:subunit interface. The C-terminal domain contains a typical Rossmann fold and orients the dinucleotide. The dimer has overall dimensions of approximately 82 A x 75 A x 75 A, with roughly 50 A separating the two active sites. The structures described here, namely the enzyme.NAD+.phenylpyruvate, and enzyme. NAD+.beta-phenylpropionate species, represent the first models for any amino acid dehydrogenase in a ternary complex. By analysis of the active-site interactions in these models, along with the currently available kinetic data, a detailed chemical mechanism has been proposed. This mechanism differs from those proposed to date in that it accounts for the inability of the amino acid dehydrogenases, in general, to function as hydroxy acid dehydrogenases.     

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.     

Primary structure of human pancreatic elastase 2 determined by sequence analysis of the cloned mRNA

A cDNA encoding elastase 2 has been cloned from a human pancreatic cDNA library. The cDNA contains a translation initiation site and a poly(A) recognition site and encodes a protein of 269 amino acids, including a proposed 16-residue signal peptide. The amino acid sequence of the deduced mature protein contains a 12-residue activation peptide containing a cysteine at residue 1 similar to that of chymotrypsin. The proposed active enzyme contains all of the characteristic active-site amino acids, including His-57, Asp-102, and Ser-195. The S1 binding pocket is bounded by Gly-216 and Ser-226, making this pocket intermediate in size between chymotrypsins and elastase 1 or protease E, consistent with the substrate specificity of elastase 2 for long-chain aliphatic or aromatic amino acids. Computer modeling studies using the amino acid sequence of elastase 2 superimposed on the X-ray structure of porcine elastase 1 suggest that a change of Gln-192 in elastase 1 to Asn-192 in elastase 2 may account for the lower catalytic efficiency of the latter enzyme. In addition, modeling studies have been conducted to attempt to identify basic amino acids in elastases which are absent in chymotrypsins, and which could account for the specific property of elastolysis. Several basic residues appear to be near the ends of the extended binding pocket of elastases which might serve to anchor the enzyme to the elastin substrate. These studies indicate that elastases 2 and elastase 1 both contain an Arg-65A as well as a basic dipeptide at 223/224 which is not present in chymotrypsins.(ABSTRACT TRUNCATED AT 250 WORDS)