Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction

The pH dependence of the reaction of various renins was investigated using sheep angiotensinogen as a substrate. Human renin showed two separate peaks, but rat and mouse Ren1 renins showed one peak with a shoulder. A comparison of the predicted subsite residues of human renin with those of rat and mouse Ren1 renins revealed that Arg82, Ser84, Thr85, Ala229, and Thr312 are unique in the human sequence. We examined the possible importance of these residues in the unique pH profile of the human renin reaction by replacing these residues with the corresponding residues of rat renin. The replacement of Ser84 of human renin with Gly changed the pH dependence of the reaction to one peak, similarly to rat and mouse Ren1 renins. Other mutant human renins kept two separate peaks, similarly to wild-type human renin. These results indicate that Ser84 of human renin contributes to the biphasic pH dependence of the renin-angiotensinogen reaction.     

Tyrosine-83 of human renin contributes to biphasic pH dependence of the renin-angiotensinogen reaction.

The pH dependence of the reaction of human renin with sheep angiotensinogen had a couple of peaks at pH 6.5 and 8.7. The renin activity at pH 8.7 was 1.4 times higher than that at pH 6.5. After the substitutions of Phe, Ala, and His for Tyr83 of human renin, the peak at pH 6.5 could be observed but the peak at pH 8.7 disappeared. At pH 6.5, these substitutions reduced the kcat of human renin to 11.1, 1.31, and 3.49% of that of wild-type renin, respectively, while their Km remained at similar levels to that of the wild type. These results indicate that Tyr83 of human renin contributes to the renin-angiotensinogen reaction at both pHs and it is essential for the catalytic reaction particularly at the basic pH.     

Intramolecularly quenched fluorogenic peptide substrates for human renin.

Six intramolecularly quenched fluorogenic peptides related to the sequences Phe8 to His13, His6 to His13, and Tyr4 to His13 of the human angiotensinogen, containing o-aminobenzoyl (Abz) and ethylenediamine dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acids residues, were synthesized by classical solution methods. The Leu-Val is the only bond of all obtained peptides that was hydrolyzed by human renin with different degrees of purity and was resistant to hydrolysis by pig renin and cathepsin D. The hydrolysis of Abz-His-Pro-Phe-His-Leu-Val-Ile-His-EDDnp by human renin was inhibited by a highly specific transition-state analog of angiotensinogen (IC50 = 7.8 x 10(-9) M), described by K. Iizuka et al. (1990, J. Med. Chem. 33, 2707-2714). Therefore, specific and sensitive substrates for the continuous assay of human renin in which as little as 70 microGU of human renin could be detected by Abz-Phe-His-Leu-Val-Ile-His-EDDnp were described. The optimal pHs of hydrolysis of the substrates were in the range 4 to 6.     

Formation of angiotensin II by tonin from partially purified human angiotensinogen

The renin substrate (angiotensinogen) has been purified from outdated human blood bank plasma. A 100-fold purification was achieved by ammonium sulphate protein fractionation and four successive chromatographic procedures. We show that tonin, a serine protease enzyme found in submaxillary glands of the rat, cleaves the human plasma angiotensinogen, devoid of tonin inhibiting factor(s), at a pH optimum of 5--5.5. It generates a pressor substance that was identified as angiotensin (A) II. The rate of cleavage of the human angiotensinogen preparation by 1 nmol of renin or tonin was calculated to be 1320 nmol AI/h for renin and 26 nmol AII/h for tonin.     

The His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin

The amino acid sequence His-Pro-Phe as N-terminal residues 6-8 of the natural renin substrate, angiotensinogen, is conserved among species. We investigated whether this His-Pro-Phe motif functions as the determinant of the substrate specificity of renin. Mutant angiotensinogens in which the Ile-His-Pro-Phe-His-Leu sequence at positions 5-10 of wild-type angiotensinogen was replaced by either His-Pro-Phe-His-Leu-Leu or Ala-Ile-His-Pro-Phe-His were cleaved by renin at the C-terminal side of residues 9 and 11, respectively, while wild-type angiotensinogen was cleaved at residue 10. A triple Ala substitution for the His-Pro-Phe motif of angiotensinogen prevented its cleavage by renin. In contrast, triple Ala substitution for residues 9-11, including the natural site of cleavage by renin, allowed cleavage between the two Ala residues at positions 10 and 11. Furthermore, the 33-residue C-terminal peptide of human megsin, which carries a naturally occurring His-Pro-Phe sequence, was cleaved by renin at the C-terminal side of the His-Pro-Phe-Leu-Phe sequence. These results indicate that the His-Pro-Phe motif of angiotensinogen is a crucial determinant of the substrate specificity of renin. By binding to a corresponding pocket on renin, the His-Pro-Phe motif may act as a molecular anchor to recruit the scissile peptide bond to a favorable site for catalysis.     

Synthesis of human angiotensinogen (1-17) containing one of the putative glycosylation binding sites and its hydrolysis by human renin and porcine pepsin.

The N-terminal heptadecapeptide of human angiotensinogen (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Asn-Glu-Ser-Thr-NH2 ), with the C-terminal carboxyl group amidated, was synthesized in order to study the role of Asn-Glu-Ser, a putative carbohydrate binding site, on the hydrolysis by human renin. The synthesis was performed by fragment condensation using the Honzl and Rudinger azide procedure. In our conditions for azide segment condensation, histidine racemization was demonstrated to be negligible for most of the condensation reactions. Human renin liberates angiotensin I from h-angiotensinogen (1-17)-NH2 with a Km value of 3.4 x 10(-5) M, at pH 7.3 and 37 degrees being similar to h-angiotensinogen (1-13), an analog without the carbohydrate binding site. However, the Vmax value of 4.1 x 10(-9) mol/G.U. min is one order of magnitude higher. Porcine pepsin was demonstrated to cleave preferentially Leu10-Val11 bond and, surprisingly, His9-Leu10 as well.     

Isorenin, pseudorenin, cathepsin D and renin. A comparative enzymatic study of angiotensin-forming enzymes

1. Renin was purified 30 000-fold from rat kidneys by chromatography on DEAE-cellulose and SP-Sephadex, and by affinity chromatography on pepstatinyl-Sepharose. 2. The enzymatic properties of isorenin from rat brain, pseudorenin from hog spleen, cathepsin D from bovine spleen, and renin from rat kidneys were compared: Isorenin, pseudorenin and cathepsin D generate angiotensin from tetradecapeptide renin substrate with pH optima around 4.9, renin at 6.0. With sheep angiotensinogen as substrate, isorenin, pseudorenin and cathepsin D have similar pH profiles (pH optima at 3.9 and 5.5), in contrast to renin (pH optimum at 6.8). 3. The angiotensin-formation from tetradecapeptide by isorenin, pseudorenin and cathepsin D was inhibited by albumin, alpha-and beta-globulins. These 3 enzymes have acid protease activity at pH 3.2 with hemoglobin as the substrate. Renin is not inhibited by proteins and has no acid protease activity. 4. Renin generates angiotensin I from various angiotensinogens at least 100 000 times faster than isorenin, pseudorenin or cathepsin D, and 3000 000 times faster than isorenin when compared at pH 7.2 with rat angiotensinogen as substrate. 5. The 3 'non-renin' enzymes exhibit a high sensitivity to inhibition by pepstatin (Ki less than 5.10(-10) M), in contrast to renin (Ki approximately 6-10(-7) M), at pH 5.5. 6. It is concluded from the data that isorenin from rat brain and pseudorenin from hog spleen are closely related to, or identical with cathepsin D.     

Comparison of angiotensinogen and tetradecapeptide as substrates for human renin. Substrate dependence of the mode of inhibition of renin by a statine-containing hexapeptide

The kinetic properties of two different substrates for human renin, a synthetic tetradecapeptide and the natural substrate human angiotensinogen, have been compared. While the Vmax was similar for the two substrates, the Km values differed by a factor of 10, i.e., 11.7 +/- 0.7 microM (tetradecapeptide) and 1.0 +/- 0.1 microM (angiotensinogen). The mode of inhibition of renin by a statine (Sta)-containing hexapeptide, BW897C, that is a close structural analog of residues 8-13 of human angiotensinogen (Phe-His-Sta-Val-Ile-His-OMe), was determined for the two substrates. Competitive inhibition was observed when tetradecapeptide was the substrate (Ki = 2.0 +/- 0.2 microM), but a more complex mixed inhibition mode (Ki = 1.7 +/- 0.1 microM, Ki' = 3.0 +/- 0.23 microM) was found with angiotensinogen as substrate. This mixed inhibition probably results from the formation of an enzyme-inhibitor-substrate or enzyme-inhibitor-product complex and reflects the more extensive interactions that the protein angiotensinogen, as opposed to the small tetradecapeptide substrate, can make with renin. We conclude that the mixed inhibition observed when angiotensinogen is used as renin substrate could be important in the clinical application of renin inhibitors because it is less readily reversed by increased concentrations of substrate than is simple competitive inhibition.     

Renin cleavage of a human kidney renin substrate analogous to human angiotensinogen, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, that is human renin specific and is resistant to cathepsin D

A synthetic tetradecapeptide, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, which corresponds to the 13 amino terminal residues of human angiotensinogen plus a carboxy terminal serine to replace a suggested site of carbohydrate attachment, has been shown to be a good substrate for human kidney renin. At pH 7.2 and 37 degrees C the KM or Michaelis constant was 8.4 +/- 2.9 microM, and the VM or velocity at infinite tetradecapeptide concentration was 11.3 +/- 2.4 mumol angiotensin I made per hour per milligram renin. The tetradecapeptide was highly resistant to cleavage by mouse submaxillary renin. The tetradecapeptide was also slowly cleaved by human liver cathepsin D, by rabbit lung angiotensin-converting enzyme, and by reconstituted human serum, but did not yield angiotensin I. Thus, this synthetic renin substrate should permit more specific measurement of human kidney renin activity.     

Functional characterization of Asp-317 mutant of human renin expressed in COS cells

Renin is an unique aspartyl (acid) protease with optimal activity at neutral pH. It has been suggested that Ala-317 of human renin contributes to neutral optimum pH of the enzyme [(1984) FEBS Lett. 174, 102–111]. The hypothesis was verified by the characterization of mutant renin in which Ala-317 was replaced with Asp by a site-directed mutagenesis. Wild-type and mutant renins, which were expressed in COS cells, exhibited different pH-activity profiles and optimum pH of the mutant enzyme was lower than that of the wild-type enzyme. This result suggests that Ala-317 of human renin plays an important role in the determination of optimum pH of the enzyme.     

Characterization of pure human renal renin. Evidence for a subunit structure

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.     

Purification, properties and kinetics of sheep and human renin substrates.

Sheep plasma renin substrate was purified 1,200-fold by using nephrectomised sheep plasma, followed by DEAE-Sephadex chromatography and gel filtration. The purified substrate contained 8 mu-g angiotensin II/mg protein and had an estimated molecular weight of 52,000. The kinetic characteristics of the purified substrate were identical both to those of unpurified nephrectomised sheep plasma and to normal sheep plasma substrates. At pH 7.5, K-m of the human renin-sheep substrate reaction was 0.29 mu-M and for sheep renin-sheep substrate, 2.0 mu-M. Sheep substrate was susceptible to peptic digestion with generation of pepsitensin. Human renin substrate was less readily purified. DEAE-Sephadex chromatography of plasma from pregnant women at 36-40 weeks' gestation produced a 70-fold increase in purity (0.9 mu-g angiotensin II/mg protein). No further increase was achieved with gel filtration. Human renin substrate behaved as a larger (mol. wt. 82,000) more anionic protein than sheep substrate and was resistant to the proteolytic actions of both pepsin and sheep renin. K-m for the human renin-human substrate reaction was high and could not be accurately determined (range 3-8 mu-M, mean 5.7 mu-M). The presence of human substrate in a human renin-sheep substrate system did not alter the measured initial velocity. In both sheep and man, the normal concentration of renin substrate is considerably less than K-m and must therefore be considered a determinant of angiotensin production rate in vivo.     

Expression of renin and angiotensinogen genes in the human placental tissues

The expression of renin and angiotensinogen genes in the human placenta and related tissues has been examined by RNA blot hybridization analysis with specific human complementary DNA (cDNA) probes. Renin mRNA was detectable in the chorion throughout pregnancy and in the hydatidiform moles, but not in the decidua, amnion or myometrium. The relative concentration of renin mRNA in the chorion was at the highest level in early pregnancy and decreased thereafter, while the total amount contained in the whole placenta was at the lowest level in early pregnancy, and increased thereafter, reaching at term about one-sixth of the total renin mRNA in the kidney. Hydatidiform moles had an even higher concentration of renin mRNA than the early chorion. There was no significant difference in either the relative concentration or the total renin mRNA content in the placentae from 4 normal and 4 toxemic pregnancies. Angiotensinogen mRNA was undetectable in any of the placental tissues, hydatidiform moles or myometrium. These results show that renin is synthesized in the placenta, possibly to play some physiological role locally by utilizing angiotensinogen which is abundantly present in the maternal systemic circulation.     

Peptide analogues of angiotensinogen. Effect of peptide chain length on renin inhibition

Renin inhibition was evaluated for a series of peptide analogues of angiotensinogen with different chain lengths. Systematic deletion of amino acid residues from the hexapeptide BocPheHisLeuR-ValIleHisOCH3 showed that the presence of residues at the N-terminal Phe and His positions was essential for efficient enzyme-inhibitor binding whereas the C-terminal Ile and His residues were much less important. Synthesis of a tetrapeptide analogue shortened at the C-terminus and containing modified side chains produced a potent inhibitor of renin which demonstrated hypotensive activity in a salt depleted monkey.     

13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase

On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2. 69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6. The Y229 mutant shows only the internal commitment of 0.6. The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.     

Assignment by in situ hybridization of the angiotensinogen gene to chromosome band 1q4, the same region as the human renin gene

Summary A 1.8kb human cDNA probe for angiotensinogen (renin substrate) was used to determine the chromosomal location of the angiotensinogen gene by in situ hybridization. The results show that human chromosome region 1q4 contains the angiotensinogen gene. The human renin gene has also recently been assigned to the same band of chromosome 1. Thus, the angiotensinogen and renin genes are located in the same region of chromosome 1.     

Comparative studies on species-specific reactivity between renin and angiotensinogen

The renin-angiotensin system (RAS) is the most important regulator of electrolyte homeostasis and blood pressure. Our recently generated transgenic mice carrying either the human renin (hREN) or human angiotensinogen (hANG) genes did not develop hypertension but dual gene strains obtained by cross-mating separate lines of mice exhibited a chronically sustained increase in blood pressure, suggesting the presence of species-specific reactivity between renin and angiotensinogen. In order to examine this specificity, the present study was designed to perform a strictly comparative study on hydrolysis of hANG by hREN and mouse submandibular renin (mREN)in vitro by using pure proteins. The recombinant hANG (rhANG) and the synthetic human-type tridecapeptide (hTDP), Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His, corresponding to the N-terminal sequences of hANG, were used to determine the species specificity of recombinant hREN (rhREN) and mREN. While hTDP was cleaved by both rhREN with similar Km and with the same order of kcat, rhANG was cleaved by mREN with 16.7-fold higher Km and with 28.2-fold lower kcat than by rhREN. These results showed that kcat/Km value of mREN for rhANG was 468-fold lower than that for rhREN acting on rhANG.     

pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase and trypsin

The pH dependency of the carboxyl oxygen exchange reaction catalyzed by lysyl endopeptidase (Lys-C) and trypsin has been studied. The reaction was quantitatively monitored by measuring the incorporation of 18O atom into the alpha-carboxyl group of N(alpha)-acetyl-L-lysine from H2(18)O solvent. The optimum pHs of the carboxyl oxygen exchange reaction catalyzed by Lys-C and trypsin were found to be pH 5.0 and 6.0, respectively, which were significantly shifted toward acidic pHs compared to the most favorable pHs of their amidase activities for N(alpha)-acetyl-L-lysine amide in the pHs examined. Steady-state kinetics parameters were also determined for both enzymes at two different pHs, one at the pH optimum for their carboxyl oxygen exchange activity (pH 5-6) and the other at the favorable pH for their amidase activity (pH 8-9). Significantly lower Km (2-fold lower for Lys-C, 3-fold lower for trypsin), and higher kcat values (1.5-fold higher for Lys-C, 5-fold higher for trypsin) were obtained at the acidic pHs compared to the alkaline pHs, suggesting that Lys-C and trypsin have higher substrate binding affinities and higher catalytic rates at the acidic pHs than at the alkaline pHs. The higher carboxyl oxygen exchange activities at the acidic pHs were also confirmed with peptide substrates derived from apomyoglobin. These findings are significant toward the goal of improving the efficiency of the Lys-C and trypsin catalyzed 18O labeling reactions and are thus pertinent to improving the accuracy and reliability of quantitative proteomic experiments utilizing 18O labeling.     

The amino-acid residues on the C-terminal side of the cleavage site of angiotensinogen influence the species specificity of reaction with renin

The N-terminal sequences of human and canine angiotensinogen and two hybrid sequences were synthesized and used to determine whether the species specificity of renin is influenced by amino-acid residues adjacent to the cleavage site. kcat/Km for the generation of angiotensin I from the N-terminal tridecapeptide of human angiotensinogen by canine renin is 0.37% of that observed when the N-terminal tetradecapeptide from canine angiotensinogen is used as a substrate. Replacement of the valine residue at P'1 in the human tridecapeptide with the leucine residue from the canine sequence triples kcat and improves Km 4-fold. Replacement of isoleucine residue at P'2 with the valine residue from the canine sequence enhances Km 8-fold. Substitution of the histidine residue at P'3 with the tyrosine serine sequence of canine angiotensinogen increases kcat an order of magnitude. Results obtained with the synthetic substrate are similar to those observed with the protein substrates. Canine renin does not cleave human angiotensinogen. Also, kcat/Km of canine renin for its homologous substrate is about 6-times greater than the kcat/Km value for human renin acting on human angiotensinogen.     

Comparison of starch hydrolysis activity and thermal stability of two Bacillus licheniformis alpha-amylases and insights into engineering alpha-amylase variants active under acidic conditions

Bacillus licheniformis alpha-amylase (BLA) is widely used in various procedures of starch degradation in the food industry, and a BLA species with improved activity at higher temperature and under acidic conditions is desirable. Two BLA species, designated as PA and MA, have been isolated from the wild-type B. licheniformis strain and a mutant strain, respectively. In this study, their starch-hydrolysis activity and thermal stability were examined. MA showed higher activity than PA, especially at acidic pH (pH 5.0-5.5), and even after 1 h of treatment at 90 degrees C. MA was active in the range of pH 4.0-8.0, which is much wider than that (pH 4.5-7.5) of PA. It was shown that the proton dissociation constants on the acidic and alkaline sides (pKa1 and pKa2) were shifted to more acidic and basic values, respectively, by the mutation of PA to MA. The activation energy and thermodynamic parameters for their thermal inactivation indicate that MA is more thermally stable and catalytically active than PA, suggesting that MA could be useful for glucose-production process coupled with reactions catalyzed by beta-amylase.