Active-site mobility in human immunodeficiency virus, type 1, protease as demonstrated by crystal structure of A28S mutant.

The mutation Ala28 to serine in human immunodeficiency virus, type 1, (HIV-1) protease introduces putative hydrogen bonds to each active-site carboxyl group. These hydrogen bonds are ubiquitous in pepsin-like eukaryotic aspartic proteases. In order to understand the significance of this difference between HIV-1 protease and homologous, eukaryotic aspartic proteases, we solved the three-dimensional structure of A28S mutant HIV-1 protease in complex with a peptidic inhibitor U-89360E. The structure has been determined to 2.0 A resolution with an R factor of 0.194. Comparison of the mutant enzyme structure with that of the wild-type HIV-1 protease bound to the same inhibitor (Hong L, Treharne A, Hartsuck JA, Foundling S, Tang J, 1996, Biochemistry 35:10627-10633) revealed double occupancy for the Ser28 hydroxyl group, which forms a hydrogen bond either to one of the oxygen atoms of the active-site carboxyl or to the carbonyl oxygen of Asp30. We also observed marked changes in orientation of the Asp25 catalytic carboxyl groups, presumably caused by the new hydrogen bonds. These observations suggest that catalytic aspartyl groups of HIV-1 protease have significant conformational flexibility unseen in eukaryotic aspartic proteases. This difference may provide an explanation for some unique catalytic properties of HIV-1 protease.     

Conserved interactions of the active carboxyls in pepsin-like enzymes and retroviral proteases

In addition to previous studies, 30 crystal structures of retroviral proteases corresponding to the highest resolution were inspected to analyze the interactions of the active carboxyl with surroundings groups. The outer oxygen of the active carboxyl in retroviral enzymes form contacts only with the water molecule participating in catalysis. This is an important difference between retroviral proteases and pepsin-like enzymes, which form a net of hydrogen bonds of these outer oxygen with residues neighboring the catalytic site in 3D structures. At the same time, it was found that in all aspartic proteases the inner oxygen of the active carboxyl are also involved in the chain of interactions through peptide groups Thr-Gly adjacent to the active residues. Polarization of these peptide groups influences the donor-acceptor properties of the active carboxyl. The found chain of interactions is more extensive in retroviral than in pepsin-like proteases; however, its main part is conserved for the whole class of these enzymes. Some implications of the role of these interactions are discussed.     

Special Features of the Three-Dimensional Structure Defining Properties of Aspartic Proteases

A brief account is given of the specific interactions of some amino acid residues in aspartic proteases of both higher organisms and retroviruses that determine important protease properties: the anomalously low isoelectric point of pepsin and its stability at pH close to 1; the ability of one of the carboxyl groups in the active site of proteases of higher organisms to retain a charged state at any pH value; and the protonated state of another carboxyl, which is necessary for enzymatic activity. It is also explained how such states can be induced in retroviral proteases.     

Crystal structure of an activation intermediate of cathepsin E

Cathepsin E is an intracellular, non-lysosomal aspartic protease expressed in a variety of cells and tissues. The protease has proposed physiological roles in antigen presentation by the MHC class II system, in the biogenesis of the vasoconstrictor peptide endothelin, and in neurodegeneration associated with brain ischemia and aging. Cathepsin E is the only A1 aspartic protease that exists as a homodimer with a disulfide bridge linking the two monomers. Like many other aspartic proteases, it is synthesized as a zymogen which is catalytically inactive towards its natural substrates at neutral pH and which auto-activates in an acidic environment. Here we report the crystal structure of an activation intermediate of human cathepsin E at 2.35A resolution. The overall structure follows the general fold of aspartic proteases of the A1 family, and the intermediate shares many features with the intermediate 2 on the proposed activation pathway of aspartic proteases like pepsin C and cathepsin D. The pro-sequence is cleaved from the protease and remains stably associated with the mature enzyme by forming the outermost sixth strand of the interdomain beta-sheet. However, different from these other aspartic proteases the pro-sequence of cathepsin E remains intact after cleavage from the mature enzyme. In addition, the active site of cathepsin E in the crystal is occupied by N-terminal amino acid residues of the mature protease in the non-primed binding site and by an artificial N-terminal extension of the pro-sequence from a neighboring molecule in the primed site. The crystal structure of the cathepsin E/pro-sequence complex, therefore, provides further insight into the activation mechanism of aspartic proteases.     

Five atomic resolution structures of endothiapepsin inhibitor complexes: implications for the aspartic proteinase mechanism

Endothiapepsin is derived from the fungus Endothia parasitica and is a member of the aspartic proteinase class of enzymes. This class of enzyme is comprised of two structurally similar lobes, each lobe contributing an aspartic acid residue to form a catalytic dyad that acts to cleave the substrate peptide bond. The three-dimensional structures of endothiapepsin bound to five transition state analogue inhibitors (H189, H256, CP-80,794, PD-129,541 and PD-130,328) have been solved at atomic resolution allowing full anisotropic modelling of each complex. The active sites of the five structures have been studied with a view to studying the catalytic mechanism of the aspartic proteinases by locating the active site protons by carboxyl bond length differences and electron density analysis. In the CP-80,794 structure there is excellent electron density for the hydrogen on the inhibitory statine hydroxyl group which forms a hydrogen bond with the inner oxygen of Asp32. The location of this proton has implications for the catalytic mechanism of the aspartic proteinases as it is consistent with the proposed mechanism in which Asp32 is the negatively charged aspartate. A number of short hydrogen bonds (approximately 2.6 A) with ESD values of around 0.01 A that may have a role in catalysis have been identified within the active site of each structure; the lengths of these bonds have been confirmed using NMR techniques. The possibility and implications of low barrier hydrogen bonds in the active site are considered.     

A range of catalytic efficiencies with avian retroviral protease subunits genetically linked to form single polypeptide chains.

Molecular modeling based on the crystal structure of the Rous sarcoma virus (RSV) protease dimer has been used to link the two identical subunits of this enzyme into a functional, single polypeptide chain resembling the nonviral aspartic proteases. Six different linkages were selected to test the importance of different interactions between the amino acids at the amino and carboxyl termini of the two subunits. These linkages were introduced into molecular clones of fused protease genes and the linked protease dimers were expressed in Escherichia coli and purified. Catalytically active proteins were obtained from the inclusion body fraction after renaturation. The linked protease dimers exhibited a 10-20-fold range in catalytic efficiencies (Vmax/Km) on peptide substrates. Both flexibility and ionic interactions in the linkage region affect catalytic efficiency. Some of the linked protease dimers were 2-3-fold more active than the nonlinked enzyme purified from bacteria, although substrate specificities were unchanged. Similar relative efficiencies were observed using a polyprotein precursor as substrate. Mutation of one catalytic Asp in the most active linked protease dimer inactivated the enzyme, demonstrating that these proteins function as single polypeptide chains rather than as multimers.     

Kinetics of the dimerization of retroviral proteases: the "fireman's grip" and dimerization

All retroviral proteases belong to the family of aspartic proteases. They are active as homodimers, each unit contributing one catalytic aspartate to the active site dyad. An important feature of all aspartic proteases is a conserved complex scaffold of hydrogen bonds supporting the active site, called the "fireman's grip," which involves the hydroxyl groups of two threonine (serine) residues in the active site Asp-Thr(Ser)-Gly triplets. It was shown previously that the fireman's grip is indispensable for the dimer stability of HIV protease. The retroviral proteases harboring Ser in their active site triplet are less active and, under natural conditions, are expressed in higher enzyme/substrate ratio than those having Asp-Thr-Gly triplet. To analyze whether this observation can be attributed to the different influence of Thr or Ser on dimerization, we prepared two pairs of the wild-type and mutant proteases from HIV and myeloblastosis-associated virus harboring either Ser or Thr in their Asp-Thr(Ser)-Gly triplet. The equilibrium dimerization constants differed by an order of magnitude within the relevant pairs. The proteases with Thr in their active site triplets were found to be approximately 10 times more thermodynamically stable. The dimer association contributes to this difference more than does the dissociation. We propose that the fireman's grip might be important in the initial phases of dimer formation to help properly orientate the two subunits of a retroviral protease. The methyl group of threonine might contribute significantly to fixing such an intermediate conformation.     

X-ray structure of HIV-1 protease in situ product complex

HIV-1 protease is an effective target for design of different types of drugs against AIDS. HIV-1 protease is also one of the few enzymes that can cleave substrates containing both proline and nonproline residues at the cleavage site. We report here the first structure of HIV-1 protease complexed with the product peptides SQNY and PIV derived by in situ cleavage of the oligopeptide substrate SQNYPIV, within the crystals. In the structure, refined against 2.0-A resolution synchrotron data, a carboxyl oxygen of SQNY is hydrogen-bonded with the N-terminal nitrogen atom of PIV. At the same time, this proline nitrogen atom does not form any hydrogen bond with catalytic aspartates. These two observations suggest that the protonation of scissile nitrogen, during peptide bond cleavage, is by a gem-hydroxyl of the tetrahedral intermediate rather than by a catalytic aspartic acid.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

嗜水气单胞菌胞外蛋白酶的化学修饰

 蛋白酶是嗜水气单胞菌 (Aeromonashydrophila)的重要致病因子 .为研究其结构与功能之间的关系 ,用DEPC、EDC、PMSF、N AI等 9种化学修饰剂处理嗜水气单胞菌J 1株胞外蛋白酶ECPase54,然后检测残余酶活力 ,借以研究酶分子中氨基酸侧链基团与酶活性中心的关系 .结果表明 ,羧基、丝氨酸、ε 氨基、胍基等残基与酶活性无关 ;半胱氨酸残基与酶活性也无直接关系 ;而色氨酸、组氨酸、酪氨酸残基侧链以及二硫键的化学修饰引起酶活性的大幅度的下降 ,说明色氨酸、组氨酸、酪氨酸残基以及二硫键是酶活力所必需的基团     

Identification of catalytically active groups of pea (Pisum sativum L.) beta-glucosidase

Functional groups ofcytoplasmic pea beta-glucosidase pretreated to an electrophoretically homogeneous state were identified. Data on the pH dependence of the enzyme activity, calculated heat of ionization, photoinactivation of the enzyme in the presence of methylene blue, and inactivation of the enzyme with diethyl pyrocarbonate suggest that the catalytic site of beta-glucosidase contains the carboxyl group of glutamic or aspartic acids and the imidazole group of histidine.     

Mutational analysis of human immunodeficiency virus type 1 protease suggests functional homology with aspartic proteinases.

Processing of the retroviral gag and pol gene products is mediated by a viral protease. Bacterial expression systems have been developed which permit genetic analysis of the human immunodeficiency virus type 1 protease as measured by cleavage of the pol protein precursor. Deletion analysis of the pol reading frame locates the sequences required to encode a protein with appropriate proteolytic activity near the left end of the pol reading frame but largely outside the gag-pol overlap region, which is at the extreme left end of pol. Most missense mutations within an 11-amino-acid domain highly conserved among retroviral proteases and with sequence similarity to the active site of aspartic proteinases abolish appropriate processing, suggesting that the retrovirus proteases share a catalytic mechanism with aspartic proteinases. Substitution of the amino acids flanking the scissile bond at three of the processing sites encoded by pol demonstrates distinct sequence requirements for cleavage at these different sites. The inclusion of a charged amino acid at the processing site blocks cleavage. A subset of these substitutions also inhibits processing at the nonmutated sites.     

Crystallographic analysis of transition state mimics bound to penicillopepsin: difluorostatine- and difluorostatone-containing peptides.

Difluorostatine- and difluorostatone-containing peptides have been evaluated as potent inhibitors of penicillopepsin, a member of the aspartic proteinase family of enzymes. Isovaleryl-Val-Val-StaF2NHCH3 [StaF2 = (S)-4-amino-2,2-difluoro-(R)-3-hydroxy-6-methylheptanoic acid] and isovaleryl-Val-Val-StoF2NHCH3 [StoF2 = (S)-4-amino-2,2-difluoro-3-oxo-6-methylheptanoic acid] have measured Ki's of 10 x 10(-9) and 1 x 10(-9) M, respectively, with this fungal proteinase. The StoF2-containing peptide binds 32-fold more tightly to the enzyme than the analogous peptide containing the non-fluorinated statine ethyl ester. Each compound was cocrystallized with penicillopepsin, intensity data were collected to 1.8-A resolution, and the atomic coordinates were refined to an R factor [formula: see text] of 0.131 for both complexes. The inhibitors bind in the active site of penicillopepsin in much the same fashion as do other statine-containing inhibitors of penicillopepsin analyzed earlier [James, M. N. G., Sielecki, A. Salituro, F., Rich, D. H., & Hofmann, T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6137-6141; James, M.N.G., Sielecki, A., & Hofmann, T. (1985) in Aspartic Proteinases and their Inhibitors (Kosta, V., Ed.) pp 163-177, Walter deGruyter, Berlin]. The (R)-3-hydroxyl group in StaF2 binds between the active site carboxyl groups of Asp33 and Asp213, making hydrogen-bonding contacts to each one. The ketone functional group of the StoF2 inhibitor is bound as a hydrated species, with the gem-diol situated between the two aspartic acid carboxyl groups in a manner similar to that predicted for the tetrahedral intermediate expected during the catalytic hydrolysis of a peptide bond [James, M. N. G., & Sielecki, A. (1985) Biochemistry 24, 3701-3713]. One hydrogen-bonding interaction from the "outer" hydroxyl group is made to O delta 1 of Asp33, and the "inner" hydroxyl group forms two hydrogen-bonding contacts, one to each of the carboxyl groups of Asp33 (O delta 2) and Asp213 (O delta 2). The only structural difference between the StaF2 and StoF2 inhibitors that accounts for the factor of 10 in their Ki's is the additional (R)-3-OH group on the tetrahedral sp3 carbon atom of the hydrated StoF2 inhibitor. The intermolecular interactions involving the fluorine atoms of each inhibitor are normal van der Waals contacts to one of the carboxyl oxygen atoms of Asp213 (F2-O delta 2 Asp213, 2.9 A). The observed stereochemistry of the bound StoF2 group in the active site of penicillopepsin has stimulated our reappraisal of the catalytic pathway for the aspartic proteinases.(ABSTRACT TRUNCATED AT 400 WORDS)     

1.9 A x-ray study shows closed flap conformation in crystals of tethered HIV-1 PR

Three-dimensional structure of an asymmetrically mutated (C95M) tethered human immunodeficiency virus type 1 protease enzyme (HIV-1 PR) has been determined in an unliganded form using X-ray diffraction data to 1.9 A resolution. The structure, refined using X-PLOR to an R factor of 19.5%, is unexpectedly similar to the ligand-bound native enzyme, rather than to the ligand-free native enzyme. In particular, the two flaps in the tethered dimer are in a closed configuration. The environments around M95 and C1095 are identical, showing no structural effect of this asymmetric mutation at position 95. Oxidation of Cys1095 has been observed for the first time. There is one well-defined water molecule that hydrogen bonds to both carboxyl groups of the essential aspartic acids in the active site. Proteins 2001;43:57-64.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.     

Identification of catalytically active groups of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) β-glucosidase

Functional groups of cytoplasmic pea β-glucosidase pretreated to an electrophoretically homogeneous state were identified. Data on the pH dependence of the enzyme activity, calculated heat of ionization, photoinactivation of the enzyme in the presence of methylene blue, and inactivation of the enzyme with diethyl pyrocarbonate suggest that the catalytic site of β-glucosidase contains the carboxyl group of glutamic or aspartic acids and the imidazole group of histidine.     

Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

The relatively fast artificial substrate Leu-Ser-rho-nitro-Phe-Nle-Ala-Leu-OMe generates a solvent isotope effect of 1.51 +/- 0.02 only on the maximal velocity of peptide hydrolysis catalyzed by porcine pepsin (EC 3.4.23.1). The absence of an isotope effect on V/K places the isotopically-sensitive step after peptide bond cleavage and the release of the first product. Reprotonation of the active site aspartic carboxyls is proposed as the most likely interpretation of this observation. Structural and kinetic similarities between pepsin and other aspartic proteinases, including the therapeutically important targets HIV protease and renin, suggest a similar slow reprotonation step after catalysis. This mechanistic feature has important implications regarding inhibitor design; if most of the enzymes are present in a product-release form during steady-state turnover, then perhaps inhibitors should be designed as product analogs instead of substrate analogs.     

Probing the S1/S1' substrate binding pocket geometry of HIV-1 protease with modified aspartic acid analogues

Aspartates 25 and 125, the active site residues of HIV-1 protease, participate functionally in proteolysis by what is believed to be a general acid-general base mechanism. However, the structural role that these residues may play in the formation and maintenance of the neighboring S1/S1' substrate binding pockets remains largely unstudied. Because the active site aspartic acids are essential for catalysis, alteration of these residues to any other naturally occurring amino acid by conventional site-directed mutagenesis renders the protease inactive, and hence impossible to characterize functionally. To investigate whether Asp-25 and Asp-125 may also play a structural role that influences substrate processing, a series of active site protease mutants has been produced in a cell-free protein synthesizing system via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The suppressor tRNAs were activated with the unnatural aspartic acid analogues erythro-beta-methylaspartic acid, threo-beta-methylaspartic acid, or beta,beta-dimethylaspartic acid. On the basis of the specific activity measurements of the mutants that were produced, the introduction of the beta-methyl moiety was found to alter protease function to varying extents depending upon its orientation. While a beta-methyl group in the erythro orientation was the least deleterious to the specific activity of the protease, a beta-methyl group in the threo orientation, present in the modified proteins containing threo-beta-methylaspartate and beta,beta-dimethylaspartate, resulted in specific activities between 0 and 45% of that of the wild type depending upon the substrate and the substituted active site position. Titration studies of pH versus specific activity and inactivation studies, using an aspartyl protease specific suicide inhibitor, demonstrated that the mutant proteases maintained bell-shaped pH profiles, as well as suicide-inhibitor susceptibilities that are characteristic of aspartyl proteases. A molecular dynamics simulation of the beta-substituted aspartates in position 25 of HIV-1 protease indicated that the threo-beta-methyl moiety may partially obstruct the adjacent S1' binding pocket, and also cause reorganization within the pocket, especially with regard to residues Val-82 and Ile-84. This finding, in conjunction with the biochemical studies, suggests that the active site aspartate residues are in proximity to the S1/S1' binding pocket and may be spatially influenced by the residues presented in these pockets upon substrate binding. It thus seems possible that the catalytic residues cooperatively interact with the residues that constitute the S1/S1' binding pockets and can be repositioned during substrate binding to orient the active site carboxylates with respect to the scissile amide bond, a process that likely affects the facility of proteolysis.     

Modeling of substrate and inhibitory complexes of histidine-aspartic protease

A three-dimensional structure of histo-aspartic protease (HAP), a pepsin-like enzyme from the causative agent of malaria Plasmodium falciparum, is suggested on the basis of homologous modeling followed by equilibration by the method of molecular dynamics. The presence of a His residue in the catalytic site instead of an Asp residue, which is characteristic of pepsin-like enzymes, and replacement of some other conserved residues in the active site make it possible for the enzyme to function by the covalent mechanism inherent in serine proteases. The detailed structures of HAP complexes with pepstatin, a noncovalent inhibitor of aspartic proteases, and phenylmethylsulfonyl fluoride, a covalent inhibitor of serine proteases, as well as with a pentapeptide substrate are discussed.