Biomedical subjects

H Umeyama

Publications and source records attributed to H Umeyama.

At least 37 records · Page 2Linked to original sources

PubMed1997

Prediction of protein side-chain conformations by principal component analysis for fixed main-chain atoms.

A method of side-chain prediction without calculating the potential function is introduced. It is based on the assumption that similar side-chain conformations have a similar structural environment around the side chains. The environment information is represented by vectors that were obtained from principle component analysis and represented by the variance of positions of main-chain atoms around side chains. This information was added to the side-chain library (rotamer library) made from X-ray structures. Side-chain conformations were constructed using this side-chain library without using potential functions. An optimal solution was determined by comparing environmental information with the backbone conformation around the side chain to be predicted and native ones in the library. The method was performed for 15 proteins whose structures were known. The result for the root-mean-square deviation between the predicted and X-ray side-chain conformations was approximately 1.5 A (the value for core residues was approximately 1.1 A) and the percentage of predicted chi 1 angles correct within 40 degrees was approximately 65% (75% for the core). The computational time was short (approximately 60 s for the prediction of proteins with 200 amino acid residues). About 70% of the side-chain conformations were constructed by location of the main-chain atoms around the central C beta atom and the average of r.m.s.d. was approximately 1.4 A (for core residues the average was approximately 1.0 A).

Animals↗

PubMed1997

Protein dynamics determined by backbone conformation and atom packing.

To study the factors determining the collective motions in thermal, conformational fluctuations of a globular protein, molecular dynamics simulations were performed with a backbone model and an atomic-level model. In the backbone model, only the C alpha atoms were explicitly treated with two types of pairwise interactions assigned between the C alpha atoms; atom-packing interactions to take into account the effect of tight atom packing in the protein interior and chain-restoring interactions to maintain the backbone around the native conformation. A quasi-harmonic method was used to decompose the overall fluctuations into independent, collective modes. The modes assigned to large conformational fluctuations showed a good correlation between the backbone and atomic-level models. From this study, it was suggested that the collective modes were motions in which a protein fluctuates, keeping the tertiary structure around the native one and avoiding backbone overlap and, hence, rough aspects of the collective modes can be derived without details of the atomic interactions. The backbone model is useful in obtaining the overall backbone motions of a protein without heavy simulations, even though the simulation starts from a poorly determined conformation of experiments and in sampling main chain conformations, from which the side chain conformations may be predicted.

Models, Chemical↗

PubMed1996

Controlling substrate preference and transglycosylation activity of neopullulanase by manipulating steric constraint and hydrophobicity in active center.

The substrate specificity and the transglycosylation activity of neopullulanase was altered by site-directed mutagenesis on the basis of information from a three-dimensional structure predicted by computer-aided molecular modeling. According to the predicted three-dimensional structure of the enzyme-substrate complex, it was most likely that Ile-358 affected the substrate preference of the enzyme. Replacing Ile-358 with Trp, which has a bulky side chain, reduced the acceptability of alpha-(1-->6)-branched oligo- and polysaccharides as substrates. The characteristics of the I358W-mutated enzyme were quite different from those of wild-type neopullulanase and rather similar to those of typical starch-saccharifying alpha-amylase. In contrast, replacing Ile-358 with Val, which has a smaller side chain, increased the preference for alpha-(1-->6)-branched oligosaccharides and pullulan as substrates. The transglycosylation activity of neopullulanase appeared to be controlled by manipulating the hydrophobicity around the attacking water molecule, which is most likely used to cleave the glucosidic linkage in the hydrolysis reaction. We predicted three residues, Tyr-377, Met-375, and Ser-422, which were located on the entrance path of the water molecule might be involved. The transglycosylation activity of neopullulanase was increased by replacing one of the three residues with more hydrophobic amino acid residues; Y377F, M375L, and S422V. In contrast, the transglycosylation activity of the enzyme was decreased by replacing Tyr-377 with hydrophilic amino acid residues, Asp or Ser.

Amino Acid Sequence↗

PubMed1996

Genetic characterization of protein C deficiency in Japanese subjects using a rapid and nonradioactive method for single-stand conformational polymorphism analysis and a model building.

We studied the molecular basis of protein C deficiency in 28 Japanese families including 4 asymptomatic families. Two showed a decreased level of function with a normal antigen concentration consistent with type II protein C deficiency and the remaining 26 showed type I deficiency with decreases in both function and antigen level. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining polymerase chain reaction (PCR) amplification and rapid nonradioactive single-strand conformational polymorphism (SSCP) analysis. The PCR-amplified fragments with aberrant migration on SSCP analysis were sequenced. We identified 11 missense mutations, 1 nonsense mutation, 2 neutral polymorphisms, 1 frameshift deletion, 1 inframe deletion, and 1 splice site mutation. We also identified two different rare mutations in the 5'-untranslated region in the protein C gene that may be responsible for the phenotype. Of these molecular defects, ten were novel. From the results of genetic analysis of 47 Japanese families with protein C deficiency reported in this and previous studies, Phe139Val and Met364Ile substitutions and a G8857 deletion were only found in Japanese subjects and seem to be a founder effect. In contrast, Arg169Trp and Val297Met substitutions, both occurring at CG dinucleotides, were commonly observed in not only Japanese but also Western populations, indicating that these are hot spots for mutation in the protein C gene. These molecular defects were found in 22 families in total, accounting for 47% of Japanese families with protein C deficiency. The structural models of the second EGF and protease domains of activated wild-type and mutant human protein C suggest a possible substrate binding exosite on two loops; one from amino acid position 349 to 357 and the other from position 385 to 388, both of which are close to each other in the three-dimensional model.

Amino Acid Sequence↗

PubMed1994

Roles of the aromatic residues conserved in the active center of Saccharomycopsis alpha-amylase for transglycosylation and hydrolysis activity.

The molecular structure of Saccharomycopsis fibuligera alpha-amylase was predicted by a homology-based modeling technique, and the amino acid residues composing the active site were displayed with color codes according to their order of conservation. We noticed two highly conserved aromatic residues located in the active center, tyrosine 83 (Y83) and tryptophan 84 (W84), and examined their roles in catalytic activity by site-directed mutagenesis. The W, leucine (L), and asparagine (N) mutants at Y83 and the L mutant at W84 showed remarkable enhancement of transglycosylation activity and complementary decreases in native hydrolysis activity. The phenylalanine (F) mutant at Y83 and the F and Y mutants at W84 only decreased hydrolysis activity. Mechanistic and kinetic studies of these mutants using a reducing-end-blocked substrate and a hydrolysis-specific substrate revealed a probable transglycosylation mechanism and critical contributions of the 83rd and 84th aromatic residues to efficient hydrolysis. Given that aromatic residues stack against the faces of sugars, we assumed that Y83 and, presumably, W84 play roles in the binding of oligosaccharide substrates through the stacking interaction and in the indirect fixation of the catalytic water molecule through hydrogen bonding with the hydroxyl of the bound substrates. Mutations to nonaromatic residues could cause slight changes in the binding topology of substrates to favor transglycosylation over hydrolysis.

Amino Acid Sequence↗

PubMed1994

Six missense mutations associated with type I and type II protein C deficiency and implications obtained from molecular modelling.

The molecular basis of protein C deficiency was studied in three type I and three type II heterozygotes. Three probands showed thrombotic complications. All the exons and intron/exon junctions of the protein C gene were studied using a strategy combining by the polymerase chain reaction (PCR) amplification, single-strand conformational polymorphism (SSCP) analysis, and DNA sequencing of the PCR-amplified fragments. Six missense mutations were identified, including three novel ones. One was located in exon II, in which the initiating translation codon (ATG) encoding for Met at position -42 was replaced by ACG encoding for Thr. The other five were located in exon IX, and included TAC(Tyr399)-->CAC(His), CCG(Pro327)-->CTG(Leu), GAC(Asp359)-->AAC(Asn) in two cases, and GGG(Gly350)-->AGG(Arg). Four of the six missense mutations occurred in CG dinucleotide. Sequence analysis of the other exons excluded additional mutations. By restriction enzyme analysis, co-segregation of the mutation with protein C deficiency was observed in four families. The other two mutations at amino acid positions -42 and 350 were also considered to be associated with protein C deficiency due to the absence of these mutations in 50 normal individuals. A structural model of the protease domain of mutant activated protein C was constructed by the chimeric modelling method, and the resultant model suggested conformational changes due to each missense mutation identified in protein C deficiency. The present data also provide some evidence regarding the genetic heterogeneity of protein C deficiency.

Adult↗

PubMed1994

Factor IX Bm Kiryu: a Val-313-to-Asp substitution in the catalytic domain results in loss of function due to a conformational change of the surface loop: evidence obtained by chimaeric modelling.

Factor IX Kiryu is a naturally occurring mutant of factor IX that has 2.5% coagulant activity, even though normal plasma levels of factor IX antigen are detected. Factor IX Kiryu was purified from a patient's plasma by immunoaffinity chromatography with a calcium-dependent anti-factor IX monoclonal antibody column. It was cleaved normally by factor XIa in the presence of Ca2+, yielding a two-chain factor IXa. However, the resulting factor IXa showed only 1.5% of the normal factor IXa in terms of factor X activation in the presence of factor VIII, phospholipids, and Ca2+, and had 20% of the normal esterase activity for Z-Arg-p-nitrobenzyl ester. Therefore factor IXa Kiryu showed the defect of the catalytic triad or primary substrate binding site as well as defective interaction with factors VIII/X. Single-strand conformational polymorphism analysis and DNA sequencing of the amplified DNA revealed a missense point mutation, a T-to-A substitution at nucleotide number 31,059 of the factor IX Kiryu gene. This mutation resulted in the amino acid substitution of Val-313 by Asp in the catalytic domain. Restriction enzyme analysis of the amplified DNA showed that the mutation was inherited from the patient's mother. The chimaeric method was employed to construct a model of the serine protease domain of factor IXa, and the resultant model suggested that the Val-313 to Asp substitution altered the conformation of the substrate-binding site. These data combined with our previous findings on a Gly-311-to-Glu mutant of factor IX suggest that the loop conformation from Gly-311 to ARg-318 is important for the expression of coagulant activity.

Amino Acid Sequence↗

PubMed1994

Three-dimensional model of the human PAF receptor.

The amino acid sequence analysis of the human platelet-activating factor (PAF) receptor showed that residues thought to be important structurally and functionally were well conserved. This suggested similarities of the three-dimensional structure (3D structure) between the human PAF receptor and other receptors that couple to guanine nucleotide binding (G) proteins. Thus, a three-dimensional model of this receptor was constructed using the 3D structure of bacteriorhodopsin as the reference protein, by means of the BIOCES[E] computer-modeling system. This model has seven alpha-helical transmembrane segments which form a central core and an S-S bond between the second and third extracellular loops. The distance of the S-S bond is about 2 A, which is thought to be reasonable. In the transmembrane domain, the side chains of Asp-63, Asn-285 and Asp-289 became oriented toward the central core and form a negatively charged site. This receptor model suggests that the positively charged choline moiety of PAF is attracted to this negatively charged site by electrostatic forces and that PAF may induce conformational changes in the receptor, leading to G-protein activation.

Amino Acid Sequence↗

PubMed1993

Specificity of the sequence in Phe-Gln-Val-Val-Cys (-3-nitro-2-pyridinesulfenyl)-Gly-NH2--a selective inhibitor of thrombin-induced platelet aggregation.

Thrombin-induced platelet aggregation is mediated in part by the intracellularly activated calpain expressed onto the external side of the membrane. We have previously shown that P1, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2 [Npys = 3-nitro-2-pyridinesulfenyl], an affinity analog corresponding to the highly conserved sequence Gln-Val-Val-Ala-Gly-NH2, present in domains 2 and 3 of human kininogens, was an irreversible inhibitor of platelet calpain (second-order rate constant = 5.85 mM-1 s-1). P1 also selectively blocked thrombin-induced platelet aggregation. We have now synthesized twenty-three other peptides, analogous to P1, and evaluated them to define the specificity of the amino acid sequence in P1 to selectively block thrombin-induced platelet aggregation. We find that replacement by Leu of Val and by Tyr of Phe adjacent to Gln is minimally tolerated and the resulting peptides are partially effective in selectively blocking thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. The presence of valine adjacent to cysteine in P1 is essential for the inhibitor to selectively block thrombin-induced platelet aggregation. Extensions of the N-terminal sequence in P1 did not improve its selectivity. Ac-Ala-Gln-Val-Val-Ala-Gly-NH2 (Ac, acetyl), a peptide containing the conserved sequence but lacking the Npys function, neither inhibited platelet calpain nor platelet aggregation induced by thrombin. Presence of the peptide sequence and Npys function are both required in P1 for its selective action in inhibiting platelet aggregation induced by thrombin.

Amino Acid Sequence↗

PubMed1993

Molecular defect in factor IX Tokyo: substitution of valine-182 by alanine at position P2' in the second cleavage site by factor XIa resulting in impaired activation.

Utilizing polymerase chain reaction and directly sequencing the amplified exon 6 of the factor IX gene derived from a mild hemophilia Bm patient, we have identified a T to C mutation at nucleotide 20,525. This point mutation predicted a Val182 to Ala substitution in the abnormal factor IX molecule, designated as factor IX Tokyo. The patient manifested a low factor IX activity and a moderately prolonged ox-brain prothrombin time but a normal factor IX antigen level in plasma. Immunopurified factor IX derived from the patient was found to have a normal molecular weight but a reduced specific activity (23% of normal). Limited proteolysis by activated factor XI or by a snake venom-derived factor X-activating enzyme was considerably delayed, indicating the presence of structural alteration(s) most probably at or near the second enzyme-cleavage site. Once activated, however, factor IXa Tokyo was able to activate factor X normally and was inactivated by antithrombin III also in a normal fashion. The structural model of factor IXa and a docking model of factor IX and activated factor VII (factor VIIa) suggested that the Val182 to Ala substitution would not affect the local conformation of the catalytic domain. This mutation would rather loosen the fitness of the molecule into the substrate-binding pocket of factor VIIa due to a shorter side chain of the Ala substitution at the P2' position of the second cleavage site.

Adolescent↗

PubMed1993

Modulation of thrombin-induced platelet aggregation by inhibition of calpain by a synthetic peptide derived from the thiol-protease inhibitory sequence of kininogens and S-(3-nitro-2-pyridinesulfenyl)-cysteine.

Thrombin-induced platelet aggregation has been suggested to play an important role in reocclusion following thrombolytic therapy of angioplasty for treatment of myocardial infarction. We previously demonstrated that aggregation of washed platelets by thrombin is accompanied by cleavage of aggregin, a putative ADP receptor, and that these events are indirectly mediated by calpain, expressed on the surface of the external membrane. High-molecular-mass kininogen (HK) contains, in its heavy chain, domain 2, which is responsible for its action as a potent inhibitor of platelet calpain. Domain 3 of the heavy chain of HK directly inhibits binding of thrombin to platelets, confounding mechanistic studies using the entire molecule. Moreover, HK, a protease of 120 kDa, is unsuitable as a potential pharmacological agent. The highly conserved sequence Gln-Val-Val-Ala-Gly, present in HK and its evolutionary precursors, the cystatins, is thought to be involved in the binding of cysteine proteases but is, itself, not inhibitory. An affinity analog, Phe-Gln-Val-Val-Cys(Npys)-Gly-NH2(Npys, 3-nitro-2-sulfenylpyridine), P1, corresponding to the thiol-protease-binding sequence in HK and containing a ligand, Npys, that can react with the free sulfhydryl group in the active site of calpain, was synthesized. P1 was an irreversible inhibitor of platelet calpain. P1 selectively inhibited thrombin-induced aggregation of washed platelets and platelets in plasma, but did not inhibit the aggregatory effects of other platelet agonists. P1 did not inhibit the amidolytic activity and coagulant activity of thrombin. Unlike HK, P1 did not inhibit binding of thrombin to washed platelets. P1 did not inhibit thrombin-induced platelet-shape change. P1 neither raised intracellular levels of cAMP nor did it interfere with the ability of thrombin to antagonize the rise in intracellular levels of cAMP induced by iloprost, an analog of prostaglandin I2. The design and synthesis of P1 could leave to the development of a new class of inhibitors that selectively block thrombin-induced platelet aggregation while sparing other functions of this pathophysiological protease and without inhibiting the action of other platelet agonists.

Amino Acid Sequence↗

PubMed1993

Active site dynamics of acyl-chymotrypsin.

The motions of water molecules, the acyl moiety, the catalytic triad, and the oxyanion binding site of acyl-chymotrypsin were studied by means of a stochastic boundary molecular dynamics simulation. A water molecule that could provide the nucleophilic OH- for the deacylation stage of the catalysis was found to be trapped between the imidazole ring of His-57 and the carbonyl carbon of the acyl group. It makes a hydrogen bond with the N epsilon 2 of His-57 and is held in place through a network of hydrogen-bonded water molecules in the active site. The water molecule was found as close as 2.8 A to the carbonyl carbon. This appears to be due to the constraints imposed by nonbonded interaction in the active site. Configurations were found in which one hydrogen of the trapped water shared a bifurcated hydrogen bond with His-57-N epsilon 2 and Ser-195-O gamma, with the water oxygen very close to the carbonyl carbon. The existence of such a water molecule suggests that large movement of the His-57 imidazole ring between positions suitable for providing general-base catalyzed assistance and for providing general-acid catalyzed assistance may not be required during the reaction. The simulation indicates that the side chains of residues involved in catalysis (i.e., His-57, Ser-195, and Asp-102) are significantly less flexible than other side chains in the protein. The 40% reduction in rms fluctuations is consistent with a comparable reduction calculated from the temperature factors obtained in the X-ray crystallographic data of gamma-chymotrypsin. The greater rigidity of active site residues seems to result from interconnected hydrogen bonding networks among the residues and between the residues and the solvent water in the active site.

Acylation↗

PubMed1993

Protein modelling using a chimera reference protein derived from exons.

Bovine pancreatic beta-trypsin (PDB ID-code: 1TPO) which is registered in the Brookhaven Protein Data Bank (PDB) consists of four exons. The results of homology searches for each exon in the PDB showed that homologous proteins were tonin (PDB ID-code: 1TON), rat mast cell protease (PDB ID-code: 3RP2__A), kallikrein A (PDB ID-code: 2PKA__B) and kallikrein A (2PKA__B) respectively. Thus, for the three-dimensional structure prediction of 1TPO, a chimera protein was constructed from the three proteins mentioned above and the 3-D structure prediction was performed using this chimera reference protein. The modelled structure of 1TPO was energetically optimized by molecular mechanics and molecular dynamics simulation and was compared with its X-ray crystal structure registered in the PDB. The root mean square deviations (r.m.s.d.) of main chain atoms and the neighbouring active site (5 A sphere from His57, Asp102 and Ser195) between the modelled structure and the X-ray structure were 1.66 and 0.94 A respectively. Porcine pancreatic elastase (PDB ID-code: 3EST) which is registered in the PDB was used as the reference protein and the modelled structure from 3EST was also compared with the X-ray data. The r.m.s.d. of main chain atoms and that of the active site were 2.14 and 1.18 A respectively. These results clearly support the propriety of this method using the chimera reference protein.

Algorithms↗

PubMed1992

Prothrombin Salakta: substitution of glutamic acid-466 by alanine reduces the fibrinogen clotting activity and the esterase activity.

Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.

Alanine↗

PubMed1992

Aggregation of washed platelets by plasminogen and plasminogen activators is mediated by plasmin and is inhibited by a synthetic peptide disulfide.

Plasmin is known to activate platelets. However, it is not clear whether plasminogen activators as used in thrombolytic therapy can aggregate platelets and how this relates to the ability of each activator to convert plasminogen to plasmin. Urokinase (UK) and streptokinase (SK) activated purified plasminogen (2 microM) in a concentration-dependent manner. The rates of aggregation of washed platelets by the above plasminogen activators and plasminogen were similar to the extent of activation of plasminogen to plasmin in the absence of platelets. UK or SK (0.2 microM) and plasminogen (2 microM) aggregated platelets modified by an ADP affinity analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA), and cleaved aggregin, a putative ADP receptor, in [3H]FSBA-modified platelets. These results suggest that the effect was independent of ADP. In contrast, incubation mixtures containing only plasminogen (2 microM) and single chain tissue plasminogen activator (sc-tPA) (less than or equal to 0.12 microM) neither activated the zymogen to an appreciable extent nor aggregated platelets. But, in the presence of fibrin(ogen) fragments (tPA-stimulator), a mixture of plasminogen and sc-tPA aggregated unmodified and FSBA-modified platelets, and cleaved aggregin. The results imply that platelets, in the presence of t-PA stimulator, potentiate activation of plasminogen to plasmin by t-PA, as previously reported. P1, Phe-Gln-Val-Val-Cys-(NpyS)-Gly-NH2, (NpyS = 3-nitro-2-thiopyridine), a synthetic hexapeptide capable of binding to and inhibiting calpain, has been shown to inhibit platelet aggregation induced by purified plasmin. P1 inhibited platelet aggregation by plasminogen and any of the three plasminogen activators. Our results show that at plasma concentrations of plasminogen and at levels of UK and SK attained after infusion of these agents during thrombolysis, these mixtures can cause maximum aggregation which may contribute to reocclusion and stenosis following infarct therapy. P1 can effectively inhibit platelet aggregation under such conditions.

Amino Acid Sequence↗

PubMed1991

Factor IX Amagasaki: a new mutation in the catalytic domain resulting in the loss of both coagulant and esterase activities.

Factor IX Amagasaki (AMG) is a naturally occurring mutant of factor IX having essentially no coagulant activity, even though normal levels of antigen are detected in plasma. Factor IX AMG was purified from the patient's plasma by immunoaffinity chromatography with an anti-factor IX monoclonal antibody column. Factor IX AMG was cleaved normally by factor VIIa-tissue factor complex, yielding a two-chain factor IXa. Amino acid composition and sequence analysis of one of the tryptic peptides isolated from factor IX AMG revealed that Gly-311 had been replaced by Glu. We identified a one-base substitution of guanine to adenine in exon VIII by amplifying exon VIII using the polymerase chain reaction method and sequencing the product. This base mutation also supported the replacement of Gly-311 by Glu. In the purified system, factor IXa AMG did not activate factor X in the presence of factor VIII, phospholipids, and Ca2+, and no esterase activity toward Z-Arg-p-nitrobenzyl ester was observed. The model building of the serine protease domain of factor IXa suggests that the Gly-311----Glu exchange would disrupt the specific conformational state in the active site environment, resulting in the substrate binding site not forming properly. This is the first report to show the experimental evidence for importance of a highly conserved Gly-142 (chymotrypsinogen numbering) located in the catalytic site of mammalian serine proteases so far known.

Amino Acid Sequence↗

PubMed1991

Prediction of octanol/water partition coefficients using parameters derived from molecular structures.

A method is presented to estimate log P values using molecular surface area, electrostatic potentials and charge transfer interactions derived from three-dimensional molecular structures. Estimated logP values for 63 small organic molecules with a variety of structures gave a correlation coefficient of 0.983 with a standard deviation of 0.260. The method is applicable to rather complex and large molecules. A striking feature of the method is that it can estimate the logP values of novel compounds to which "fragment constant approaches" have not been applicable so far.

Chemical Phenomena↗

PubMed1990

Protein modeling of human prorenin using the molecular dynamics method.

To study the activation-inactivation mechanism of the renin zymogen, prorenin, a tertiary structural model of human prorenin was constructed using computer graphics and molecular dynamics calculations, based on the pepsinogen structure. This prorenin model shows that the folded prosegment polypeptide can fit into the substrate binding cleft of the renin moiety. The three positively charged residues, Arg10, Arg15, and Arg20, in the prosegment make salt bridges with Asp225, Glu331, and Asp60, respectively, in renin. Arg43, which is in the processing site, forms salt bridges with the catalytic residues of Asp81 and Asp269. These ionic interactions between the prosegment and the renin may contribute to keeping the prorenin structure as an inactive form.

Amino Acid Sequence↗