Substrate binding properties of converting enzyme using a series of p-nitrophenylalanyl derivatives of angiotensin I.

The binding properties of angiotensin I for the active site of rabbit lung converting enzyme (CE) have been investigated. A series of angiotensin I like substrates, all containing the C-terminal tripeptide, (NO2)Phe-His-Leu, were synthesized by increasing the length of the peptide at the N-terminal end. A total of eight peptides were studied, the largest being [Asn1, (NO2)Phe8]angiotensin I. As determined by thin-layer chromatography, all substrates were hydrolyzed only at the (NO2)Phe-His bond by purified converting enzyme, with the release of the dipeptide, His-Leu. By using an absorbance increase upon hydrolysis, the Michaelis constants and velocity maxima were determined and used to estimate those amino acids in the angiotensin I molecule that contribute significantly to binding to converting enzyme. It was hypothesized that, upon addition or substitution of one or more amino acids to the N-terminal end, a proportional decrease in both KM and Vm is needed in order to conclude that the substrate actually increases its affinity for the enzyme. A test of the proportionality for the variation of KM and Vm was found to be positive for all the substrates, except the N-terminal carbobenzoxy-blocked tripeptide, Z(NO2)Phe-His-Leu. Substitutions near the bond that is hydrolyzed (e.g., proline for the carbobenzoxy group) appear to alter the catalytic properties of CE, while additions far removed from the site of hydrolysis (e.g., the N-terminal tripeptide Asn-Arg-Val) may enhance binding affinity.     

Pulmonary angiotensin-converting enzyme antienzyme antibody.

A method has been developed for quantitating anticatalytic activity in antibody preparations made in goats against pure solubilized angiotensin-converting enzyme from rabbit pulmonary membranes. Anticatalytic activity was purified about 90-fold from a single batch of serum by a procedure including diethylaminoethylcellulose chromatography and elution from Sepharose columns containing covalently bound pure enzyme. Antiholoenzyme antibody was fractionated with respect to charge and binding affinity; however, these different populations each inhibited enzymatic hydrolysis of hippurylhistidylleucine, angiotensin I, and bradykinin. The inhibition dose-response curves were similar for hydrolysis of hippurylhistidylleucine and angiotensin I despite the difference in molecular weight of these substrates. Evidence is presented suggesting that a single molecule of antibody can bind two molecules of enzyme and that at least 18% of the total antiholoenzyme antibody population is directed against determinants which influence catalytic activity. A competitive immunoassay was developed with radioiodinated pulmonary enzyme as displaceable antigen. The anticatalytic and radioimmune assays were used to examine immunological properties of converting enzymes in various rabbit organs and fluids. Kidney, brain, and serum were found to contain converting enzymes which were immunologically identified with that in rabbit lung. Converting enzyme in seminal plasma was similar to the lung enzyme in the anticatalytic assay, but showed lower immunoreactivity in the radioimmune assay.     

A new feature of angiotensin-converting enzyme in the brain: hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.     

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.     

Sbustrate specificity of the elastase and the chymotrypsin-like enzyme of the human granulocyte.

Human granulocyte elastase (EC 3.4.21.11) differs from hog pancreatic elastase in its specificity for synthetic substrates. Although hydrolyzing peptide bonds adjacent to the carboxyl group of alanine, the granulocyte enzyme prefers valine at the cleaved bond, in contrast to the pancreatic enzyme which prefers alanine. Peptide bonds involving the carboxyl group of isoleucine can be hydrolyzed by the granulocyte enzyme but are not hydrolyzed to any significant extent extent by pancreatic elastase. This difference in specificty could explain the lower sensitivity of the granulocyte enzyme to inhibitors containing alanine analogs, such as the peptide chloromethyl ketones and elastatinal. The human granulocyte chymotrypsin-like enzyme differs from pancreatic chymotrypsin by being able to cleave substrates containing leucine in addition to those containing the aromatic amino acids.     

Purification and Characterization of a Peptidyl Dipeptidase Resembling Angiotensin Converting Enzyme from the Electric Organ of Torpedo marmorata

The electric organ of Torpedo marmorata contains a membrane-bound, captopril-sensitive metallopeptidase that resembles mammalian angiotensin converting enzyme (peptidyl dipeptidase A; EC 3.4.15.1). The Torpedo enzyme has now been purified to apparent homogeneity from electric organ by a procedure involving affinity chromatography using the selective inhibitor lisinopril immobilised to Sepharose via a 28-A spacer arm. The purified protein, like the mammalian enzyme, acted as a peptidyl dipeptidase in cleaving dipeptides from the C-terminus of a variety of peptide substrates, including angiotensin I, bradykinin, [Met5]enkephalin, [Leu5]enkephalin, and the model substrate hippuryl (benzoylglycyl; BzGly)-His-Leu. The hydrolysis of BzGly-His-Leu was activated by Cl-. Enzyme activity was inhibited by classical angiotensin converting enzyme inhibitors, including captopril, enalaprilat (MK422), and lisinopril (MK521). Torpedo angiotensin converting enzyme, like its mammalian counterpart, was also able to act as an endopeptidase in hydrolysing the amidated neuropeptide substance P. Hydrolysis of substance P occurred primarily at the Phe8-Gly9 bond with release of the C-terminal tripeptide, Gly-Leu-MetNH2, and this hydrolysis was blocked by selective inhibitors. The Torpedo enzyme was recognised by a polyclonal antibody to pig kidney angiotensin converting enzyme on immunoelectrophoretic (Western) blot analysis. Thus, on the basis of substrate specificity, inhibitor sensitivity, and immunological criteria, the Torpedo enzyme closely resembles mammalian angiotensin converting enzyme. However, the Torpedo enzyme appears somewhat larger (Mr = 190,000) than the pig kidney enzyme (Mr = 180,000) on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The endogenous peptide substrate(s) for Torpedo electric organ angiotensin converting enzyme and the physiological role of the enzyme in this tissue remain to be evaluated.     

Evaluation of a method for determining the free carboxyl group distribution in pectins

The distribution of free carboxyl groups in pectins has been investigated by a method that involves blocking the free carboxyl groups by glycolation, and hydrolysis of the methylesterified regions with a mixture of pectic enzymes. The hydrolysis products are separated from the glycolated regions on an ion exchange column and after deglycolation the oligomer size distribution is obtained by Sephacryl S-200 chromatography.The method was applied to five pectins with degrees of esterification in the range 5–70%. For two of the samples (an enzyme and an alkali de-esterified low methoxyl pectin) the degree of hydrolysis was significantly lower than would be predicted from the initial degree of esterification and thus for these materials the values obtained for the carboxyl group block sizes were considered to be a maximum rather than an accurate estimate.All the samples investigated had a significant proportion of free carboxyl regions with a degree of polymerisation greater than 10. With the possible exception of the pectate (degree of esterification 5%) none of the samples had a random distribution of carboxyl groups. This was considered to be a reflection of the distribution in the native pectin rather than indicating that chemical de-esterification was non-random. The large free carboxyl group block sizes was consistent with the egg-box model for low methoxyl pectin gelation. Larger blocks were found in the enzyme de-esterified pectin compared with the alkali and acid de-esterified material.     

Some Characteristics of Hydrolysis of Synthetic Substrates and Proteins by the Alkaline Proteinase from Aspergillus sojae

The specificity of the alkaline proteinase from Aspergillus sojae was investigated. In the specificity studies with synthetic substrates, the enzyme hydrolyzed the peptide linkages involving the carboxyl group of leucine, tyrosine, phenylalanine, arginine and lysine. In the hydrolysis of natural proteins, the enzyme liberated relatively large peptides and traces of free amino acids, suggesting that the enzyme is of a typical endo-type.

N- and C-Terminal amino acid residues appearing during time course digestion of various proteins were determined. Considering the influence of amino acid composition of substrates on the frequencies of appearance of the terminal amino acids, it was estimated that the susceptibility of peptide bonds of substrate to the enzyme depends mainly on the carboxyl side residues, and, to far less extent, on the amino side residues of the peptide bonds. The enzyme showed relatively high specificity for lysine, tyrosine, histidine, arginine and phenylalanine residues at the carboxyl side of the susceptible linkages.     

Studies on the N-acetyl-beta-D-hexosaminidase B from germinating Lupinus luteus L. seeds. II. Mechanism and inhibition with some 2-acetamido-2-deoxyaldono(1----4)lactones

The N-acetyl-beta-D-hexosaminidase B of germinating yellow lupin seeds catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and -galactosaminide substrates. The investigation of the pH dependence of the kinetic parameters (Vmax and Vmax/Km) demonstrated that two common ionizable groups (probably two carboxyl groups) play an essential role in the catalysis. That is, the enzyme has a lysozyme-like splitting mechanism, and the possibility of an anchimeric assistance provided by the acetamido group seems to be negligible. The presence of a deprotonated carboxyl group near the glycosidic linkage was also supported by inhibition with 1-thio substrate analogues. On the other hand, some 2-acetamido-2-deoxyaldono(1----4)lactones proved to be effective inhibitors of the hexosaminidase with the exception of the D-arabinose derivative, which can be explained by high stereospecificity in the binding.     

Conversion of atriopeptin II to atriopeptin I by atrial dipeptidyl carboxy hydrolase

We are examining the substrate specificity of atrial dipeptidyl carboxyhydrolase, a membrane-bound metallo enzyme that we isolated from bovine atrial tissue homogenates. This enzyme readily removes the dipeptide, Phe-Arg, from Bz-Gly-Ser-Phe-Arg, a stand-in substrate for atriopeptin II, one of several atrial natriuretic factors. We now report that the atrial enzyme cleaves the C-terminal dipeptide, Phe-Arg, from atriopeptin II to form atriopeptin I. The km (pH 7.5) is 25 microM and the ratio of relative Vmax/km as a measure of substrate specificity indicates that atriopeptin II is a 240-fold better substrate than Bz-Gly-His-Leu. Only Phe-Arg was detected as a hydrolysis product, indicating that sequential cleavage of Asn-Ser from atriopeptin II does not occur, and that atriopeptin I is not a substrate. Bz-Gly-Asn-Ser was as good a substrate for the atrial enzyme as Bz-Gly-His-Leu, but Bz-Cys(bzl)-Asn-Ser was not hydrolyzed. This result suggests that the presence of an intact disulfide bond or an S-alkylated residue in the P1 position of a substrate (as in atriopeptin I) prevents hydrolysis by the atrial enzyme. Comparative studies were made with the angiotensin I converting enzyme. Atriopeptin II was not a substrate. The stand-in substrates for atriopeptin I, Bz-Cys(bzl)-Asn-Ser and Bz-Gly-Asn-Ser were barely hydrolyzed, which by itself suggests that atriopeptin I is not a substrate of the angiotensin converting enzyme. Our results strongly suggest that atriopeptin II is converted to atriopeptin I and that hydrolysis is mediated by the atrial enzyme. The angiotensin I converting enzyme plays no role in processing these peptides. We suggest that the atrial enzyme be named atrial peptide convertase.     

Active Center and Mode of Reaction of Blasticidin S Deaminase

Blasticidin S deaminase (EC 3.5.4.23) was purified by affinity chromatography using a column of Sepharose 4B coupled with pyrimidinoblasticidin S as a ligand. The Michaelis constant Km and the maximum velocity F_max varied with pH, which suggests that enzyme ionization is important either for substrate binding or for catalytic activity. The inflection point at pH 8.6 ~ 8.9 in the plot of pKm versus pH was attributed to an enzyme amino group which corresponded to the carboxyl group of substrates, and an inflection at pH 5.3 in the log V_max-pH plot was assigned to the imidazole group of histidine, which appeared to be critical for catalytic deaminohydroxylation. The binding of substrates by the enzyme was inferred to be promoted thermodynamically, and activation of the substrate-enzyme complex was presumed to proceed endothermically. From the results obtained, a hypothetical mode of reaction for blasticidin S deaminase is proposed.     

Phytase from Wheat Bran

myo-Inositol mono-, di-, tri-, tetra-, and pentaphosphate were prepared by enzymic hydrolysis of myo-inositol hexaphosphate with a 1,500-fold purified phytase preparation from wheat bran and the subsequent Dowex 1 column chromatography. Relative initial rates of hydrolysis of these inositol phosphates by phytase were nearly the same each other and the activation energy of hydrolysis was about 11,000 cal. per mole for all these substrates. Km values did not vary widely with the substrates. The hydrolysis of inositol phosphates proceeded in a complicated way, except inositol monophosphate, where the reaction was of the first order. The enzyme hydrolyzed the substrates in the manner that removed phosphate group of them one by one. When mixed substrate was used the enzyme showed a preferential attack on the highest member of the phosphates present. From the mixed substrate test, it was concluded that wheat bran phytase is a single enzyme.     

Angiotensin-converting enzyme from human tissues. Physicochemical, catalytic, and immunological properties

Angiotensin-converting enzyme was purified from human lung, kidney, testis, blood plasma, and seminal plasma using a facile two-step protocol which included affinity chromatography on Sepharose-bound lisinopril followed by either gel filtration or hydroxylapatite chromatography. Molecular mass for converting enzyme from all sources except testis was 140 kDa. That from testis consisted of both a 90- and a 140-kDa form in a 4:1 ratio. Detergent-extracted membrane-bound converting enzyme aggregated on gel filtration chromatography, while trypsin-extracted and soluble converting enzyme did not. Comparison of detergent-extracted and trypsin-extracted membrane-bound converting enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing indicated that the membrane binding sequence contributed minimally to the size and charge of the enzyme. Catalytic and kinetic properties assessed by interaction with substrates, inhibitors, and anti-converting enzyme immunoglobulin were similar for all forms and sources of converting enzyme. Enzyme-linked immunosorbent assay revealed only partial homology between the 90- and 140-kDa forms of the enzyme.     

Purification and characterization of paralytic shellfish toxin-transforming enzyme, sulfocarbamoylase I, from the Japanese bivalve Peronidia venulosa

The Japanese bivalve Peronidia venulosa contains paralytic shellfish toxin (PST)-transforming enzymes that convert the weakly toxic C-toxins to the more potent decarbamoyl toxins. The enzyme was purified 154-fold with a yield of 0.26% and was named sulfocarbamoylase I. It was found to be a protein with an estimated molecular weight of 300 kDa by gel filtration column chromatography. Observation of a single band equivalent to 150 kDa on SDS-PAGE with or without reducing agents suggested it to be a homodimer with ionically bound subunits. The enzyme catalyzes the hydrolysis of the carboxyl bond in the N-sulfocarbamoyl moiety of PSP-toxins. The sulfonyl moiety in the carbamoyl side chain of substrates is essential for enzyme recognition. The N-terminal amino acid sequences of nine tryptic peptides were determined by the Edman degradation method. In a database search using the BLAST program, no protein that shows remarkable homology was retrieved. Several characteristics of the enzyme were also compared with those of another PST-transforming enzyme, carbamoylase I, which was previously isolated from the Japanese clam Mactra chinensis.     

Purification and characterization of endo-beta-galactosidase from Escherichia freundii induced by hog gastric mucin

A new procedure for inducing and purifying endo-beta-galactosidase from Escherichia freundii was described. The enzyme was found to be induced with high efficiency in culture medium containing Smith-degraded hog gastric mucin, which was prepared from a commercially available starting material. Endo-beta-galactosidase was then purified by ammonium sulfate fractionation, DEAE-Sephadex chromatography, and affinity chromatography on Sepharose conjugated with the Smith-degraded mucin. The enzyme thus purified by only three steps showed no other glycosidase or protease activities and had higher specific activity compared to the previous method. This new method has a great advantage since the gastric mucin is abundantly available and the efficiency of enzyme production was high without significant induction of exoglycosidase. The hydrolysis of oligosaccharides, glycosphingolipid, and keratansulfate was studied by using this newly purified enzyme. Kinetic data indicate that hydrolyzability of these substrates is largely affected by substrate concentration, enzyme concentration and the structure of substrates. Based on these results, the specificity of E. freundii endo-beta-galactosidase was discussed.     

Glycosylasparaginase activity requires the alpha-carboxyl group, but not the alpha-amino group, on N(4)-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N(4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free alpha-carboxyl group and a free alpha-amino group; computational studies suggest that the alpha-amino group actively participates in the catalytic mechanism. In order to study the importance of the alpha-carboxyl group and the alpha-amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (DeltaDeltaGb) for those analogues that were substrates was calculated. The results show that the alpha-amino group may be substituted with a group of comparable size, for the alpha-amino group contributes little, if any, to the transition state binding energy of the natural substrate. The alpha-amino group position acts as an "anchor" in the binding site for the substrate. On the other hand, the alpha-carboxyl group is necessary for enzyme activity; removal of the alpha-carboxyl group or changing it to an alpha-carboxamide group results in no hydrolysis reaction. Also, N-acetyl-D-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the alpha-amino group in the catalytic mechanism as indicated in computational studies.     

Chemical modification of horseradish peroxidase. Preparation and characterization of tracer enzymes with different isoelectric points.

Horseradish peroxidase (HRP), a plant glycoprotein with a molecular weight of 40,000 D and a molecular radius (ae) of 30 A, has been modified chemically to prepare tracer molecules with different molecular charge. Modification of free carboxyl groups on the enzyme is achieved by carbodiimide activation and subsequent reaction of activated carboxyl groups with a nucleophile; uncharged groups or radicals containing additional positively charged moieties are introduced into the protein molecule resulting in an increased net positive charge of the tracer. Amino groups in the protein molecule are modified by acetylation or succinylation; this reaction will increase the net negative charge of the enzyme by either introducing an uncharged group or an additional carboxyl radical. The tracer molecules so obtained are then characterized in terms of molecular size and charge by column chromatography and isoelectric focusing respectively. The enzymatic activity as measured by 3,3'-diaminobenzidine reaction, the pH optimum and the absorption spectra for the modified enzymes remain virtually unchanged.     

Chemical conversion of aspartyl peptides to isoaspartyl peptides. A method for generating new methyl-accepting substrates for the erythrocyte D-aspartyl/L-isoaspartyl protein methyltransferase

Mammalian protein carboxyl methyltransferases have recently been proposed to recognize atypical configurations of aspartic acid and may possibly function in the metabolism of covalently altered cellular proteins. Consistent with this proposal, the tetrapeptide tetragastrin, containing a single "normal" L-aspartyl residue (L-Trp-L-Met-L-Asp-L-Phe-NH2) was found here not to be an in vitro substrate for erythrocyte carboxyl methyltransferase activity. However, chemical treatment of tetragastrin by methyl esterification and then de-esterification of the aspartic acid residue yielded a mixture of peptide products, the major one of which could now be enzymatically methylated. We show here that this new peptide species is the isomeric beta-aspartyl form of tetragastrin (L-iso-tetragastrin; L-Trp-L-Met-L-Asp-L-Phe-NH2), and it appears that isomerization proceeds via an intramolecular succinimide intermediate during the de-esterification procedure. L-iso-Tetragastrin is stoichiometrically methylated (up to 90% in these experiments) with a Km for the enzyme of 5.0 microM. Similar chemical treatment of several other L-aspartyl peptides also resulted in the formation of new methyltransferase substrates. This general method for converting normal aspartyl peptides to isoaspartyl peptides may have application in the reverse process as well.     

A processing enzyme for prorenin in mouse submandibular gland. Purification and characterization

Renin is produced from an inactive precursor, prorenin, through proteolytic cleavage at paired basic amino acid residues. In this study, an enzyme which specifically cleaves mouse Ren 2 prorenin at the paired basic residues has been purified from mouse submandibular gland by CM-Toyopearl chromatography, antipain-Sepharose chromatography, and isoelectric focusing. This enzyme, named prorenin converting enzyme, consists of two polypeptide chains of 17 and 10 kDa. The enzyme has an isoelectric point of 9.5-9.8, and its pH optimum is between 7.5 and 8.5. It specifically cleaves the peptide bond on the carboxyl side of the Arg at the Lys-Arg pair of mouse Ren 2 prorenin to yield mature renin but does not cleave mouse Ren 1 and human prorenins. Studies on the effects of inhibitors indicate that this enzyme is a serine protease that differs from the enzymes processing other prohormones at paired basic amino acid residues.     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.