Purification and Characterization of a Basic Amino Acid-Specific Peptidase from Seeds of Jack Bean (Canavalia ensiformis)

A peptidase was purified from seeds of Canavalia ensiformis by extraction with water, ammonium sulfate precipitation, and successive chromatographies on DEAE-Toyopearl 650M, butyl-Toyopearl 650M, and G-3000 SW columns. The enzyme has an apparent molecular weight of 41,000. Activity is maximal at pH 9 and 60°C. The enzyme hydrolyzed synthetic substrates at Arg-X and Lys-X bonds more rapidly than bovine trypsin did, and did not cleave protein or ester substrates. The enzyme was inhibited by alkylamines and several serine protease inhibitors such as diisopropylfluorophosphate, chymostatin, leupeptin, and benzamidine. Cysteine protease-, metalloprotease-, and proteinous trypsin inhibitors were ineffective. Inhibition by alkylamines was dependent on length of the alkyl chains. From the substrate specificity and susceptibility to chemicals, the enzyme is a unique peptidase with trypsin-like specificity.     

Detection of hydrolytic activity of trypsin with a fluorescence-chymotryptic peptide on a TLC plate

To find a new trypsin-like enzyme, a simple assay method of the hydrolysis activity for trypsin has been found. We used 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) in the peptide labeling as a substrate for the trypsin-like peptidase in this study. The peptidase activity of trypsin was detected by using an AQC-chymotryptic peptide (AHP1) obtained from bovine hemoglobin. This showed that the substrate specificity of trypsin-like peptidase was distinguishable from that of the others by this procedure, and the method was used extensively in cases of various trypsin inhibitors with no significant interference from the concomitant.     

Purification,properties and substrate specificity of a digestive trypsin from Periplaneta americana (Dictyoptera) adults

A digestive trypsin from the American cockroach (Periplaneta americana, Dictyoptera) males was purified by a combination of anionic chromatographies in low and high pressure systems. The yield was 70% with a final specific activity of 2,000 units per mg protein (substrate: benzoyl-Arg-p-nitroanilide, BRpNA). Chemical modification with TLCK (k(obs)=3.3 M(-1) s(-1); stoichiometry 1:1) and PMSF (k(obs)=0.18 M(-1) s(-1); stoichiometry 1:1) confirmed that this peptidase is a trypsin. This enzyme has a molecular weight of 29 kDa (SDS-PAGE), a pI of 6.0 and a pH optimum of 8.9. Kinetic parameters using different colorimetric, fluorimetric and internally-quenched substrates indicated that P. americana trypsin prefers to hydrolyze synthetic substrates containing more than one amino acid residue and with an arginine residue at P1 position and a hydrophobic residue at P2. This enzyme presented a Km of 120 microM for BRpNA and is competitively inhibited by benzamidine (Ki=0.25 microM). Soybean trypsin inhibitor is a tight-binding inhibitor presenting a K(D) of 0.4 nM. Differences in substrate specificity and in the reactivity of the trypsin active site groups can be related to adaptation of insects to different hosts. P. americana trypsin is an excellent model for comparison as a basal group on evolutionary studies of insect trypsins.     

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.     

Substrate specificity determination of mouse implantation serine proteinase and human kallikrein-related peptidase 6 by phage display

We constructed a random library of hexapeptides displayed on the surface of bacteriophage T7 to determine the substrate specificity of proteinases. The phage-displayed library was subjected to repeated rounds of biopanning with native implantation serine proteinase and recombinant human kallikrein-related peptidase 6 (KLK6) followed by selection and identification of putative substrates. For both enzymes, the results obtained demonstrate a preference for arginine and lysine at multiple positions in the recognition cleavage motif, confirming their previously reported trypsin-like substrate specificity. In the case of KLK6, there is also a pronounced presence of tryptophan within the cleaved peptide sequences, indicating its potential dual substrate specificity, acting as both a trypsin and chymotrypsin-like enzyme.     

Multicatalytic, High-Mr Endopeptidase from Postmortem Human Brain

The main high molecular weight (650K) multicatalytic endopeptidase has been purified from postmortem human cerebral cortex. As in other tissues and species, this enzyme is composed of several subunits of 24-31K and has three distinct catalytic activities, as shown by the hydrolysis of the fluorogenic tripeptide substrates glutaryl-Gly-Gly-Phe-7-amido-4-methylcoumarin, benzyloxycarboxyl-Gly-Gly-Arg-7-amido-4-methylcoumarin, and benzyloxycarboxyl-Leu-Leu-Glu-2-naphthylamide with hydrophobic (Phe), basic (Arg), and acidic (Glu) residues in the P1 position, respectively. These activities are distinguishable by their differential sensitivity to peptidase inhibitors. The enzyme hydrolysed neuropeptides at pH 7.4 at multiple sites with widely differing rates, ranging from 113 nmol/min/mg for substance-P, down to 2 nmol/min/mg for bradykinin. The enzyme also had proteinase activity as shown by the hydrolysis of casein. For the hydrolysis of the Tyr5-Gly6 bond in luteinizing hormone-releasing hormone, the Km was 0.95 mM and the specificity constant (kcat/Km) was 4.7 X 10(3) M-1 s-1. The bond specificity of the enzyme at neutral pH was determined by identifying the degradation products of 15 naturally occurring peptide sequences. The bonds most susceptible to hydrolysis had a hydrophobic residue at P1 and either a small (e.g., -Gly or -NH2) or hydrophobic residue at P'1. Hydrolysis of -Glu-X bonds (most notably in neuropeptide Y) and the Arg6-Arg7 bond in dynorphin peptides was also seen. Thus the three activities identified with fluorogenic substrates appear to be expressed against oligopeptides.     

The isolation of a peptidyl dipeptidase from mouse brain cytosol that cleaves adrenocorticotropic hormone-(7-10) and des-tyrosine-enkephalins

An enzyme present in mouse brain cytosol cleaves C-terminal dipeptides from substrates including ACTH-(7-10) (Phe-Arg-Trp-Gly), and des-Tyr-[Met]- and des-Tyr-[Leu]enkephalin. By means of ion-exchange chromatography and gel filtration, the peptidase was purified to a specific activity of 1570 times that of brain homogenate. At this purification, a second peptidase, which hydrolyzes Trp-Gly and other peptides [M. E. A. Reith and A. Neidle (1979) Biochem. Biophys. Res. Commun. 90, 794-800] was still present, but could be removed by preparative polyacrylamide gel electrophoresis. The des Tyr-enkephalin-cleaving enzyme has a molecular weight of about 85,000 and a pH optimum of 7.8. It is inhibited by metal-chelating and sulfhydryl reagents. The enzyme has a strong preference for substrates with an aromatic residue in the position adjacent to the C-terminal amino acid, although some peptides meeting this criterion were competitive inhibitors rather than substrates. Peptides with less than four residues were inactive and, in general, tetrapeptides were found to be more reactive than larger analogs, when peptides with common C-terminal sequences were compared. The peptidyl dipeptidase, which has not been described previously, can be readily distinguished from angiotensin-converting enzyme (EC 3.4.15.1) and from neutral endopeptidase (EC 3.4.24.11) by its subcellular localization, substrate specificity, and response to inhibitors. It was suggested that peptidyl dipeptidase-B (PDP-B, EC 3.4.15.-) would be an appropriate name for the enzyme. PDP-B is widely distributed among mouse tissues.     

The purification and characterisation of novel dipeptidyl peptidase IV-like activity from bovine serum

The discovery of a potentially novel proline-specific peptidase from bovine serum is presented which is capable of cleaving the dipeptidyl peptidase IV (DPIV) substrate Gly-Pro-MCA. The enzyme was isolated and purified with the use of Phenyl Sepharose Hydrophobic Interaction, Sephacryl S-300 Gel Filtration, and Q-Sephacryl Anion Exchange, producing an overall purification factor of 257. SDS PAGE resulted in a monomeric molecular mass of 158kDa while size exclusion chromatography generated a native molecular mass of 328kDa. The enzyme remained active over a broad pH range with a distinct preference for a neutral pH range of 7-8.5. Chromatofocusing and isoelectric focusing (IEF) revealed the enzyme's isoelectric point to be 4.74. DPIV-like activity was not inhibited by serine protease inhibitors but was by the metallo-protease inhibitors, the phenanthrolines. The enzyme was also partially inhibited by bestatin. Substrate specificity studies proved that the enzyme is capable of sequential cleavage of bovine beta-Casomorphin and Substance P. The peptidase cleaved the standard DPIV substrate, Gly-Pro-MCA with a K(M) of 38.4 microM, while Lys-Pro-MCA was hydrolysed with a K(M) of 103 microM. The DPIV-like activity was specifically inhibited by both Diprotin A and B, non-competitively, generating a K(i) of 1.4 x 10(-4) M for both inhibitors. Ile-Thiazolidide and Ile-Pyrrolidide both inhibited competitively with an inhibition constant of 3.7 x 10(-7) and 7.5 x 10(-7) M, respectively. It is concluded that bovine serum DPIV-like activity share many biochemical properties with DPIV and DPIV-like enzymes but not exclusively, suggesting that the purified peptidase may play an important novel role in bioactive oligopeptide degradation.     

A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator

A second collagenolytic serine protease has been isolated from the hepatopancreas of the fiddler crab, Uca pugilator. This enzyme cleaves the native triple helix of collagen under physiological conditions of pH, temperature, and ionic strength. In addition to its collagenolytic activity, the enzyme exhibits endopeptidase activity toward other polypeptides and small molecular weight synthetic substrates. The polypeptide bond specificity of this enzyme is similar to that of bovine trypsin as is its interaction with specific protease inhibitors. The amino-terminal sequence of this enzyme displays significant homology with other serine proteases, most notably with that of crayfish trypsin, and demonstrates that this enzyme is a member of the trypsin family of serine endopeptidases. The relatively unique action of this protease with regard to both collagenous and noncollagenous substrates has important implications concerning the specificity and mechanism of collagen degradation.     

Novel Ca2+-activated neutral protease from an aquatic fungus, Allomyces arbuscula.

A Ca2+-activated neutral protease was purified to homogeneity from an aquatic Phycomycete fungus, Allomyces arbuscula. It requires millimolar concentrations of Ca2+ for activation (1.8 to 2 mM for 50% activation). Sr2+ can replace Ca2+ but at higher concentrations (4 mM for 50% activation). The enzyme is a dimer of 40-kilodalton subunits and contains six cysteine residues, three of which are revealed only after the addition of micromolar concentrations of Ca2+; the other three are free. Enzyme activity is strongly inhibited by SH-group inhibitors and some trypsin inhibitors (leupeptin and alpha-N-tosyl-L-lysine chloromethyl ketone). The enzyme lacks general trypsinlike specificity, since substrates containing tryptic cleavage sites are not cleaved nor is enzyme activity inhibited by other trypsin inhibitors. The enzyme has many functional similarities to the extensively characterized mammalian and avian Ca2+-activated neutral proteases but differs in its substrate specificity, inhibition by alpha-N-tosyl-L-phenylalanine chloromethyl ketone, and subunit structure. It is, nevertheless, presumed that this enzyme has a similar high order of specificity and is involved in the regulation of a specific growth function.     

Characterization of endopeptidase activity of tripeptidyl peptidase-I/CLN2 protein which is deficient in classical late infantile neuronal ceroid lipofuscinosis

Endopeptidase activities of the CLN2 gene product (Cln2p)/tripeptidyl peptidase I (TPP-I), purified from rat spleen, were studied using the synthetic fluorogenic substrates. We designed and constructed decapeptides, based on the known sequence cleavage specificities of bacterial pepstatin-insensitive carboxyl proteases (BPICP). MOCAc-Gly-Lys-Pro-Ile-Pro-Phe-Phe-Arg-Leu-Lys(Dnp)r-NH(2) is readily hydrolyzed by Cln2p/TPP-I (K(cat)/K(m) = 7.8 s(-1) mM(-1)). The enzyme had a maximal activity at pH 3.0 for an endopeptidase substrate, but at pH 4.5 with respect to tripeptidyl peptidase activity. Both endopeptidase and tripeptidyl peptidase activities were strongly inhibited by Ala-Ala-Phe-CH(2)Cl, but not inhibited by tyrostatin, an inhibitor of bacterial pepstatin-insensitive carboxyl proteases, pepstatin, or inhibitors of serine proteases. Fibroblasts from classical late infantile neuronal ceroid lipofuscinosis patients have less than 5% of the normal tripeptidyl peptidase activity and pepstatin-insensitive endopeptidase activity. Cln2p/TPP-I is a unique enzyme with both tripeptidyl peptidase and endopeptidase activities for certain substrate specificity.     

The purification and specificity of a neutral endopeptidase from rabbit kidney brush border

1. A neutral peptidase, previously shown to be located in the brush border of the proximal tubule, and assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain was purified from rabbit kidney. 2. The starting material for the purification was a microsomal pellet prepared from a homogenate of cortical tissue. The membrane-bound enzymes were solubilized by treatment with toluene and trypsin. About half the neutral peptidase activity was released by this treatment in a form that no longer sedimented with the microsomal pellet and which penetrated polyacrylamide gels when subjected to disc electrophoresis. Other treatments with detergents or proteolytic enzymes either inactivated the peptidase or failed to convert it into a genuinely soluble form. 3. Chromatography with successive columns of Sephadex G-200, DEAE-cellulose and hydroxyl-apatite yielded an enzyme that was free of other brush-border peptidase activities and which was homogeneous on disc electrophoresis and ultracentrifugation. 4. The purified enzyme attacked [(125)I]iodoglucagon at a rate comparable with that for [(125)I]iodoinsulin B chain. It did not appear to attack proteins (insulin, albumin and casein) that had been similarly iodinated. 5. Unlabelled insulin B chain and unlabelled glucagon were substantially hydrolysed by the endopeptidase, whereas insulin and albumin released only trivial amounts of ninhydrin-reacting material. The resistance of insulin to attack by endopeptidase, even after prolonged incubation, was confirmed by biological and immunoassay. 6. The specificity of the peptidase was determined by analysis of the products after incubating unlabelled insulin B chain, and some oligopeptide substrates, including pentagastrin, with the enzyme. All of the bonds readily cleaved were those involving the alpha-amino group of hydrophobic residues, i.e. x-Leu-, x-Val-, x-Tyr-, x-Phe- and x-Met-, provided that the residues were not C-terminal. 7. The enzyme showed only endopeptidase activity. Substrates suitable for aminopeptidases, carboxypeptidases or esterases were not attacked.     

Purification and characterization of active recombinant rat kallikrein rK9.

The rat tissue kallikrein rK9 is most abundant in the submandibular gland and the prostate. It has been successfully expressed in the Pichia pastoris yeast expression system. A full-length cDNA coding for the mature rK9 was fused in frame with yeast alpha-factor cDNA. The fusion protein was secreted into the medium with high yield without being processed by the yeast KEX2 signal peptidase. Mature rK9 was efficiently released from the fusion protein by trypsin and was purified to homogeneity by one-step affinity chromatography using soya bean trypsin inhibitor (SBTI) as affinity ligand. The identity of the recombinant enzyme was checked by N-terminal amino acid sequencing, Western blot analysis and kinetic studies. The dual trypsin- and chymotrypsin-like enzymatic specificity of rK9 was assessed by determining specificity constants (k(cat)/K(m)) for the hydrolysis of fluorogenic substrates, the peptide sequences of which were derived from proparathyroid hormone (pro-PTH) and from semenogelin-I. Our results confirmed the presence of an extended binding site in the rK9 active site. We also identified a far more sensitive substrate of this enzyme than those previously described, Abz-VKKRSARQ-EDDnp, which was hydrolysed with a catalytic efficiency k(cat)/K(m) of 420000 M(-1)s(-1). Finally, we showed that four of the five major proteins contained in secretions of rat seminal vesicles were rapidly degraded by recombinant rK9.     

Characterization of leucine amino peptidase from Streptomyces gedanensis and its applications for protein hydrolysis

The aim of this work was to purify and characterize the extra-cellular leucine amino peptidase (LAP) from Streptomyces gedanensis and also study its applications for protein hydrolysis. The enzyme was purified to homogeneity by ammonium sulfate fractionation and sequential chromatography steps. LAP appeared to be a monomeric enzyme with a molecular weight of ～75 kDa determined by sodium dodecyl sulfate poly acryl amide gel electrophoresis (SDS-PAGE). The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Met, Phe, Lys and Arg derivatives. Kinetic studies on the purified enzyme confirmed that it can hydrolyze peptide as well as ester substrates at comparable rates. This amino peptidase was highly resistant to different concentrations of various organic solvents. The characteristics of this amino peptidase, including thermo stability, organic solvent resistance, its activity against various substrates, and also it showed esterase and peptidase activity at comparable rates; identified this amino peptidase as a novel one. The specificity towards aromatic and hydrophobic amino acid residues, the solvent-resistance and thermo stability make this amino peptidase could offer interesting possibilities for various industrial applications including debittering of protein hydrolysates, peptide and ester synthesis.     

Inhibition of Prolyl Oligopeptidase by Fmoc-Aminoacylpyrrolidine-2-Nitriles

Abstract: Prolyl oligopeptidase (EC 3.4.21.26), a widely distributed cytosolic enzyme, cleaves peptidylprolyl peptide and peptidylprolyl amino acid bonds in many neuropeptide substrates. Its action on vasopressin has been proposed as the underlying mechanism accounting for the ability of inhibitors of prolyl oligopeptidase to reverse scopolamine-induced amnesia in rats. Future behavioral studies would be facilitated by the availability of potent inhibitors readily synthesized from common intermediates. A series of Fmoc-aminoacylpyrrolidine-2-nitriles prepared by a simple two-step synthesis were found to be potent noncompetitive inhibitors of the rabbit brain enzyme. The most potent inhibitors, Fmoc-prolyl-pyrrolidine-2-nitrile and Fmoc-alanyl-pyrrolidine-2-nitrile, each have a K _i of 5 n M . The compounds are cell permeable and stable. They do not inhibit the related enzyme dipeptidyl peptidase IV (EC 3.4.14.5). When administered intraperitoneally to mice, Fmoc-alanyl-pyrrolidine-2-nitrile crosses the blood-brain barrier to inhibit brain prolyl oligopeptidase. The ease of synthesis, potency, efficacy in vivo, stability, and specificity of Fmoc-aminoacylpyrrolidine-2-nitriles may make them inhibitors of choice in studies probing the physiological significance of prolyl oligopeptidase.     

Trypsin-like protease from soybean seeds. Purification and some properties

An enzyme was purified from soybean seeds mainly by repeated ion-exchange chromatography using benzoyl-L-arginine p-nitroanilide (BAPA) as a substrate. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The molecular weight was estimated as 59,000 by gel filtration. The enzyme was most active toward BAPA between pH 8 and 10. The enzyme was inactive toward protein substrates but hydrolyzed synthetic substrates and oligopeptides exclusively at the carboxyl side of L-arginine and L-lysine. Kinetic studies using synthetic substrates showed that, on the basis of Vmax/Km, the enzyme preferentially hydrolyzed amide substrates over ester substrates. Benzoyl-L-arginine 4-methylcoumaryl-7-amide (Bz-Arg-MCA) was the best substrate. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP), tosyl-L-lysine chloromethyl ketone (Tos-Lys-CH2Cl), leupeptin, and antipain. p-Chloromercuribenzoate (PCMB) was only partially inhibitory. Various protein inhibitors of trypsin such as soybean trypsin inhibitor were ineffective. From the primary specificity and susceptibility to chemicals, the enzyme can be said to be a trypsin-like serine protease. Although the physiological role of the enzyme is unclear, it seems likely that it is involved in limited hydrolysis of certain physiological peptides during processing.     

Isolation and primary characterization of an amidase from Rhodococcus rhodochrous

Amidase (EC 3.5.1.4) was purified to homogeneity from Rhodococcus rhodochrous M8 using isopropanol fractionation and exchange chromatography on Mono Q. The isolated amidase consists of four identical subunits with molecular weight 42+/-2 kD. The activity of the enzyme is maximal at 55-60 degrees C and within the pH range 5-8. The amidase from R. rhodochrous M8 is highly sensitive to such sulfhydryl reagents as Hg2+ and Cu2+. Chelators (EDTA and o-phenanthroline) and serine proteinase inhibitors (PMSF and DIFP) did not inhibit the activity of the enzyme. The enzyme exhibits hydrolytic and acyl transferase activity and does not possess urease activity. Aliphatic amides (acetamide and propionamide) were the best substrates for the amidase from R. rhodochrous M8, whereas bulky aromatic amides were poor substrates of this enzyme. The properties of the isolated enzyme are similar to those found in the corresponding amidase from Arthrobacter sp. J-1 and an amidase with wide substrate specificity from Brevibacterium sp. R312.     

Kinetics and specificity of serine proteases in peptide synthesis catalyzed in organic solvents

Initial rates of peptide-bond synthesis catalyzed by poly(ethylene glycol)-modified chymotrypsin in benzene were determined using high-performance liquid chromatography. Enzymatic synthesis of N-benzoyl-L-tyrosyl-L-phenylalanine amide from N-benzoyl-L-tyrosine ethyl ester and L-phenylalanine amide was found to obey Michaelis-Menten kinetics an to be consistent with a ping-pong mechanism modified by a hydrolytic branch. The catalytic activity of modified chymotrypsin was dependent on both water concentration and type of organic solvent, the highest synthesis rate being obtained in toluene. Since the chymotrypsin specificity in the organic phase was actually altered, the enzyme's apparent kinetic parameters were determined for different substrates and compared to those obtained with other serine proteases in benzene. Both N-benzoyl-L-tyrosine ethyl ester and N-alpha-benzoyl-L-lysine methyl ester were comparable acyl donors in benzene and the (kcat/Km)app value of modified chymotrypsin was only 10-fold smaller than that obtained with poly(ethylene glycol)-modified trypsin in the synthesis of N-alpha-benzoyl-L-lysyl-L-phenylalanine amide. The change in chymotrypsin specificity was also confirmed through the binding of trypsin inhibitors in benzene. The overall results suggest that hydrophobic bonding between the enzyme and its substrate should not be taken into account during catalysis in the organic phase. In general, if hydrophobic interactions are involved in the binding of substrates to the active site in aqueous media, the replacement of water by hydrophobic solvents will induce some change in enzyme specificity. Moreover, secondary residues of enzyme-binding sites may also exert a significant influence on specificity since, as observed in this study, chymotrypsin exhibited high affinity for cationic substrates and cationic inhibitors as well in apolar solvents.     

The bifunctional NadR regulator of Salmonella typhimurium: Location of regions involved with DNA binding, nucleotide transport and intramolecular communication

Abstract Alcohol dehydrogenase ADH2 was purified twice from Candida guilliermondii strain A80-03, by ion exchange column chromatography on DEAE-Toyopearl 650M. The enzyme was a dimer of M _r 98 500. ADH2 had a broad substrate specificity, oxidizing secondary alcohols as well as primary alcohols. The enzyme was sensitive to several inhibitors, such as metal chelators and thiol reagents. Kinetic studies suggested that ADH2 oxidized ethanol by an iso ordered sequential mechanism.     

Selective alteration of substrate specificity by replacement of aspartic acid-189 with lysine in the binding pocket of trypsin

To test the role of Asp-189 which is located at the base of the substrate binding pocket in determining the specificity of trypsin toward basic substrates, this residue was replaced with a lysine residue by site-directed mutagenesis. Both rat trypsinogen and Lys-189 trypsinogen were expressed and secreted into the periplasmic space of Escherichia coli. The proteins were purified to homogeneity and activated by porcine enterokinase, and their catalytic activities were determined on natural and synthetic substrates. Lys-189 trypsin displayed no catalytic activity toward arginyl and lysyl substrates. Further, there was no compensatory change in specificity toward acidic substrates; no cleavage of aspartyl or glutamyl bonds was detected. Additional studies of substrate specificity involving gas-phase sequence analyses of digested natural substrates revealed an inherent but low chymotrypsin-like activity of trypsin. This activity was retained but modified by the Asp to Lys change at position 189. In addition to hydrolyzing phenylalanyl and tyrosyl peptide bonds, the mutant enzyme has the unique property of cleaving leucyl bonds. On the basis of computer graphic modeling studies of the Lys-189 side chain, it appears that the positively charged NH2 group is directed outside the substrate binding pocket. The resulting hydrophobic cavity may explain the altered substrate specificity of the mutant enzyme. The relatively low chymotrypsin-like activity of both recombinant enzymes may be due to distorted positioning of the scissile bond with respect to the catalytic triad rather than to the lack of sufficient interaction between the hydrophobic side chains and the substrate binding pocket of the enzyme.