Wilmington, DE 19880-0328 Communicated by John D. Roberts, September 10, 1990 ABSTRACT A single-chain tethered dimer of human immunodeficiency virus protease (HIV-PR) was produced by expression of a synthetic gene in Escherichia coli. The tethered dimer, which consists of two 99-amino acid HIV-PR subunits linked together by a pentapeptide, was isolated from inclusion
Mutations of human immunodeficiency virus type 1 (HIV-1) protease at four positions, Val82, Asp30, Gly48, and Lys45 were analyzed for the resulting effects on kinetics and inhibition. In these mutants, Val82 was substituted separately by Asn, Glu, Ala, Ser, Asp, and Gln; Asp30 was individually substituted by Phe or Trp; Gly48 by His, Asp, and Tyr, respectively; and Lys45 by Glu. By examination of the inhibition of a single inhibitor, the differences in Ki values between the native and mutant enzymes can range from very large to insignificant even for the mutants with substitutions at the same position. By examination of a single mutant enzyme, the same broad range of Ki changes was observed for a group of inhibitors: Thus, how much the inhibition changes from the wild-type enzyme to a mutant is dependent on both the mutation and the inhibitor. The examination of Ki changes of inhibitors with closely related structures binding to Val82 mutants also reveals that the change of inhibition involves subsites in which Val82 is not in direct contact, indicating a considerable flexibility of the conformation of HIV protease. For the catalytic activities of the mutants, the kcat and Km values of many Val82 mutants and a Lys45 mutant are comparable to the native enzyme. Surprisingly, Gly48 mutations produce enzymes with catalytic efficiency superior to that of the wild-type enzyme by as much as 10-fold. Modeling of the structure of the mutants suggests that the high catalytic efficiency of some substrates is related to an increase of rigidity of the flap region of the mutants. The examination of the relative changes of inhibition and catalysis of mutants suggests that some of the Val82 and Gly48 mutants are potential resistance mutants. However, the resistance is specific with respect to individual inhibitors.
The conformation of the synthetic renin inhibitor CP‐69,799, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X‐ray diffraction at 1.8 A resolution and refined to the crystallographic R factor of 16%. CP‐69,799 is an oligopeptide transition‐‐state analogue inhibitor that contains a new dipeptide isostere at the P1‐P1′ position. This dipeptide isostere is a nitrogen analogue of the well‐explored hydroxyethylene dipeptide isostere, wherein the tetrahedral P1′ C alpha atom has been replaced by trigonal nitrogen. The inhibitor binds in the extended conformation, filling S4 to S3′ pockets, with hydroxyl group of the P1 residue positioned symmetrically between the two catalytic aspartates of the enzyme. Interactions between the inhibitor and the enzyme include 12 hydrogen bonds and extensive van der Waals contacts in all the pockets, except for S3′. The crystal structure reveals a bifurcated orientation of the P2 histidine side chain and an interesting relative rotation of the P3 phenyl ring to accommodate the cyclohexyl side chain at P1. The binding of the inhibitor to the enzyme, while producing no large distortions in the enzyme active site cleft, results in small but significant change in the relative orientation of the two endothiapepsin domains. This structural change may represent the action effected by the proteinase as it distorts its substrate towards the transition state for proteolytic cleavage.
Crystal structures of the protease of human immunodeficiency virus type 1 (HIV-1) and two mutant proteases, V82D and V82N, have been determined. In all three cases the enzyme forms a complex with the peptidic inhibitor U-89360E. All structures have been determined to 2.3 Å resolution and have satisfactory agreement factors: 0.173 for wild type, 0.175 for V82D, and 0.182 for V82N. Comparison of the three crystal structures provides explanations which are consistent with the known kinetic properties of these mutant enzymes with the U-89360E inhibitor [
The structure of a crystal complex of recombinant human immunodeficiency virus type 1 (HIV-1) protease with a peptide-mimetic inhibitor containing a dihydroxyethylene isostere insert replacing the scissile bond has been de- The bound inhibitor diastereomer has the R configurations at both of the hydroxyl chiral carbon atoms. One of the diol hydroxyl groups is positioned such that it forms hydrogen bonds with both the active site aspartates, whereas the other interacts with only one of them. Comparison of this X-ray structure with a model-built structure of the inhibitor, published earlier, reveals similar positioning of the backbone atoms and of the side-chain atoms in the P2-P2' region, where the interaction with the protein is strongest. However, the X-ray structure and the model differ considerably in the location of the P3 and P3' end groups, and also in the positioning of the second of the two central hydroxyl groups. Reconstruction of the central portion of the model revealed the source of the hydroxyl discrepancy, which, when corrected, provided a PI-PI' geometry very close to that seen in the X-ray structure.
The crystal structures of endothiapepsin, a fungal aspartic proteinase (EC 3.4.23.6), cocrystallized with two oligopeptide renin inhibitors, PD125967 and PD125754, have been determined at 2.0-A resolution and refined to R-factors of 0.143 and 0.153, respectively. These inhibitors, which are of the hydroxyethylene and statine types, respectively, possess a cyclohexylalanine side chain at P1 and have interesting functionalities at the P3 position which, until now, have not been subjected to crystallographic analysis. PD125967 has a bis(1-naphthylmethyl)acetyl residue at P3, and PD125754 possesses a hydroxyethylene analogue of the P3-P2 peptide bond for proteolytic stability. The structures reveal that the S3 pocket accommodates one naphthyl ring with conformational changes of the Asp 77 and Asp 114 side chains, the other naphthyl group residing in the S4 region. The P3-P2 hydroxyethylene analogue of PD125754 forms a hydrogen bond with the NH of Thr 219, thereby making the same interaction with the enzyme as the equivalent peptide groups of all inhibitors studied so far. The absence of side chains at the P2 and P1' positions of this inhibitor allows water molecules to occupy the respective pockets in the complex. The relative potencies of PD125967 and PD125754 for endothiapepsin are consistent with the changes in solvent-accessible area which take place on inhibitor binding.
The conformation of a synthetic polypeptide inhibitor, bound to the active site of the fungal aspartic proteinase endothiapepsin (EC 3.4.23.6), has been determined by X-ray diffraction at 0.20-nm resolution and refined to an agreement factor of 0.20. The inhibitor:is based on a chromogenic substrate of pepsin (EC 3.4.23.1). It has, in place of the scissile bond, a reduced peptide group which is resistant to hydrolysis and mimics the tetrahedral transition state. The inhibitor binds in an extended conformation with the reduced bond close to the essential aspartate side-chains of the enzyme. The hydrogen bonds and hydrophobic interactions between the enzyme and the inhibitor do not induce large conformational changes.The aspartic proteinases, a family of enzymes including pepsin, are characterized by two essential aspartate residues (32 and 215 in pepsin) which have been shown by X-ray diffraction to reside at the active site [l -41. Pepsin is the major digestive enzyme of the stomach and like most aspartic proteinases is optimally active at low pH. Analysis of the kinetic parameters of hydrolysis, by pepsin, of a single peptide bond in small synthetic substrates has shown that the rate constants depend not only on the nature of the residues adjacent to the scissile bond, but also on several residues each side of the dipeptidyl unit indicating that aspartic proteinases have up to seven specificity subsites [5, 61. A study of the specificity of porcine pepsin [7] for tripeptides of the type : benzyloxycarbonyl-His-Xaa-XaaOMe showed that the highest k,,,lK, is obtained when both Xaa residues are phenylalanines. Varying the Xaas causes k,,, to change much more than K , indicating that tight binding at these subsites lowers the activation barrier of hydrolysis by stabilization of the transition state. A survey of the amino acids found to occur in the vicinity of pepsin-susceptible bonds in proteins [8] led to the design and synthesis of a useful chromogenic substrate nicknamed 'Ralph' based on a PhePhe dipeptide [9], Tables 1, 2.The substrate has been converted to a potent pepsin inhibitor by replacing the scissile bond with a reduced peptide group -CH2-NH-giving H-256 [4, 101 (Tables 1, 2) which inhibits endothiapepsin and porcine pepsin with Ki values of 60 nM and 40 nM respectively.
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