An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase.

In an attempt to understand the structural reasons for differences in specificity and activity of proteinases from two retroviruses encoded by human immunodeficiency virus (HIV) and myeloblastosis associated virus (MAV), we mutated five key residues predicted to form part of the enzyme subsites S1, S2 and S3 in the substrate binding cleft of the wild-type MAV proteinase wMAV PR. These were changed to the residues occupying a similar or identical position in the HIV-1 enzyme. The resultant mutated MAV proteinase (mMAV PR) exhibits increased enzymatic activity, altered substrate specificity, a substantially changed pH activity profile and a higher pH stability close to that observed in the HIV-1 PR. This dramatic alteration of MAV PR activity achieved by site-directed mutagenesis suggests that we have identified the amino acid residues contributing substantially to the differences between MAV and HIV-1 proteinases.     

Hydrolysis of synthetic chromogenic substrates by HIV-1 and HIV-2 proteinases

Kinetic constants (Km,Kcat) are derived for the hydrolysis of a number of chromogenic peptide substrates by the aspartic proteinase from HIV-2. The effect of systematic replacement of the P2 residue on substrate hydrolysis by HIV-1 and HIV-2 proteinases is examined.     

Kinetic and modeling studies of S3-S3' subsites of HIV proteinases.

Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.     

Studies on the role of the S4 substrate binding site of HIV proteinases

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.     

Effect of substrate residues on the P2' preference of retroviral proteinases.

The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.     

Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases.

Human T-cell leukemia virus type-1 (HTLV-1) is associated with a number of human diseases. Based on the therapeutic success of human immunodeficiency virus type 1 (HIV-1) PR inhibitors, the proteinase (PR) of HTLV-1 is a potential target for chemotherapy. To facilitate the design of potent inhibitors, the subsite specificity of HTLV-1 PR was characterized and compared to that of HIV-1 PR. Two sets of substrates were used that contained single amino-acid substitutions in peptides representing naturally occurring cleavage sites in HIV-1 and HTLV-1. The original HIV-1 matrix/capsid cleavage site substrate and most of its substituted peptides were not hydrolyzed by the HTLV-1 enzyme, except for those with hydrophobic residues at the P4 and P2 positions. On the other hand, most of the peptides representing the HTLV-1 capsid/nucleocapsid cleavage site were substrates of both enzymes. A large difference in the specificity of HTLV-1 and HIV-1 proteinases was demonstrated by kinetic measurements, particularly with regard to the S4 and S2 subsites, whereas the S1 subsite appeared to be more conserved. A molecular model of the HTLV-1 PR in complex with this substrate was built, based on the crystal structure of the S9 mutant of Rous sarcoma virus PR, in order to understand the molecular basis of the enzyme specificity. Based on the kinetics of shortened analogs of the HTLV-1 substrate and on analysis of the modeled complex of HTLV-1 PR with substrate, the substrate binding site of the HTLV-1 PR appeared to be more extended than that of HIV-1 PR. Kinetic results also suggested that the cleavage site between the capsid and nucleocapsid protein of HTLV-1 is evolutionarily optimized for rapid hydrolysis.     

Protein products of the rat kallikrein gene family. Substrate specificities of kallikrein rK2 (tonin) and kallikrein rK9.

Two closely related kallikrein-like proteinases having little activity toward the standard synthetic amide substrates of tissue kallikreins were isolated from the rat submandibular gland. They were found to be the protein products of the rKlk2 (tonin) and the rKlk9 genes by amino acid sequence analysis (nomenclature of the genes and proteins of the kallikrein family is according to the proposal of the discussion panel from the participants of the KININ '91 meeting held Sept. 8-14, 1991, in Munich, Germany). These two proteinases of similar structure also had very similar physicochemical properties. They differed from other kallikrein-related proteinases in having high pHi values of 6.20 (rK2) and 6.85 (rK9). Kallikrein rK2 was purified as a single peptide chain, whereas rK9 appeared as a two-chain protein after reduction. Their enzymatic properties were also very similar and differed significantly from those of other rat kallikrein-related proteinases. Unlike the five other kallikrein-related proteinases we have purified so far, kallikrein rK9 was not inhibited by aprotinin. rK9 also differed from rK2 by its tissue localization. The prostate gland contained only rK9 where it was the major kallikrein-like component. The amino acids preferentially accommodated by the proteinase S3 to S2' subsites were identified using synthetic amide and protein substrates. Unlike other kallikrein-related proteinases, rK2 had a prevalent chymotrypsin-like specificity, whereas rK9 had both chymotrypsin-like and trypsin-like properties. Both rK2 and rK9 preferred a prolyl residue in position P2 of the substrate and did not accommodate bulky and hydrophobic residues at that position, as did most of the other kallikrein-related proteinases. This P2-proline-directed specificity is necessary for processing the precursors of several biologically active peptides. Subsites accommodating residues COOH-terminal to the scissile bond were also important in determining the overall substrate specificity of these proteinases. rK2 and rK9 both showed a preference for hydrophobic residues in P2'. Other subsites upstream of the S3 subsite were found to intervene in substrate binding and hydrolysis. The restricted specificity of rK2 and rK9 is consistent with the presence of an extended substrate binding site, and hence with a processing enzyme function. Their P1 specificities enabled both proteinases to release angiotensin II from angiotensinogen and from angiotensinogen I, but rK9 was at least 100 times less active than rK2 on both substrates. The substrate specificities of rK2 and rK9 were correlated with key amino acids defining their substrate binding site.(ABSTRACT TRUNCATED AT 400 WORDS)     

S-Sulfocysteine as a Source of the Sulfur Atom of Cephalosporin C

S. lignicolum acid proteinases A-1, A-2, and B (S-PI-insensitive) were examined for the structure of the active sites by the method of Nakatani using PAD as a probe. We attempted to screen for substances that release zinc(II)-PAD from the complex of zinc(II)-PAD-S-PI-insensitive acid proteinases. Angiotensin I released zinc(II)-PAD from the complex as well as from the complex of S-PI-sensitive acid proteinases. Angiotensin I is a good substrate for these enzymes regardless of S-PI-sensitivity. These results suggest that the two carboxyl groups capable of binding to zinc(II)-PAD are at the active site regions of S. lignicolum enzymes and that they are involved in their catalytic actions.     

Substrate shape determines specificity of recognition for HIV-1 protease: analysis of crystal structures of six substrate complexes

The homodimeric HIV-1 protease is the target of some of the most effective antiviral AIDS therapy, as it facilitates viral maturation by cleaving ten asymmetric and nonhomologous sequences in the Gag and Pol polyproteins. Since the specificity of this enzyme is not easily determined from the sequences of these cleavage sites alone, we solved the crystal structures of complexes of an inactive variant (D25N) of HIV-1 protease with six peptides that correspond to the natural substrate cleavage sites. When the protease binds to its substrate and buries nearly 1000 A2 of surface area, the symmetry of the protease is broken, yet most internal hydrogen bonds and waters are conserved. However, no substrate side chain hydrogen bond is conserved. Specificity of HIV-1 protease appears to be determined by an asymmetric shape rather than a particular amino acid sequence.     

Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates.

Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.     

Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. The prtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates that prtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of alpha(s1)-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.     

Intracellular distribution of neutral proteinases and inhibitors in pig leucocytes. Isolation of two inhibitors of neutral proteinases.

Granule and post-granular-supernatant fractions were obtained from pig leucocyte cells by differential centrifugation in 0.34 M sucrose. Granule extract possesses proteinase activity at acid and at neutral pH. Three groups of neutral and a group of acid proteinases were isolated from granule extracts by chromatography on DEAE-cellulose. In the first group are present elastase-like and plasminogen-activator proteinases, that are inhibited by diisopropylphosphorofluoridate, alpha1-antitrypsin, intracellular leucocyte inhibitor and partly with p-aminomethylbenzoic acid and Trasylol. The second group of neutral proteinases is unstable under the conditions of isolation used the third group of neutral proteinases comprises collagenases that are inhibited by ethylenediamine tetraacetic acid disodium salt, alpha1-antitrypsin and leucocyte inhibitor. The acid proteinases are inhibited only with pepstatin, up to 90%. In the post-granular supernatant was found the acid proteinase activity towards hemoglobin and casein, and non-stable neutral proteolytic activity towards bovine serum albumin and serum gamma globulin. In the post-granular supernatant also the inhibitors of neutral proteinases were found. By gel filtration on Sephadex G-100 and ion-exchange chromatography on CM-cellulose two inhibitors of neutral proteinases were isolated. The majority of the inhibitor capacity (about 80%) of post-granular supernatant was eluted together with ovalbumin (Mr 43000) and the remainder with cytochrome c (12300). These inhibitors inhibit the granule neutral proteinases, acting on all substrates used, but do not inhibit granule acid proteinase. Inhibition effects of post-granular-supernatant inhibitors on trypsin and chymotrypsin were obtained only when bovine serum albumin was used as substrate. Inhibitors of post-granular supernatant are stable at pH 6-8, but unstable in the pH rnage 2-5 and are thermolabile.     

Substrate analog inhibitors of HIV-1 protease containing phenylnorstatine as a transition state element

Substrates of HIV-1 protease are classified into three groups (A, B and C) based on the amino acid residues present at P1' and P2' sites. Replacement of the scissile amide bond by phenylnorstatine in representative substrate analog sequences from class A, B and C, yielded inhibitors of HIV-1 protease. Of the twelve inhibitors synthesized in this series, class C substrate analog inhibitors are more potent inhibitors (Ki's 3.3-24 microM) than either class A or class B inhibitors. In this series of inhibitors, the (2S,3S) isomer of phenylnorstatine is preferred over the other isomers as a "transition state element" for design of inhibitors of HIV-1 protease.     

Mechanism of inhibition of the retroviral protease by a Rous sarcoma virus peptide substrate representing the cleavage site between the gag p2 and p10 proteins.

The activity of the avian myeloblastosis virus (AMV) or the human immunodeficiency virus type 1 (HIV-1) protease on peptide substrates which represent cleavage sites found in the gag and gag-pol polyproteins of Rous sarcoma virus (RSV) and HIV-1 has been analyzed. Each protease efficiently processed cleavage site substrates found in their cognate polyprotein precursors. Additionally, in some instances heterologous activity was detected. The catalytic efficiency of the RSV protease on cognate substrates varied by as much as 30-fold. The least efficiently processed substrate, p2-p10, represents the cleavage site between the RSV p2 and p10 proteins. This peptide was inhibitory to the AMV as well as the HIV-1 and HIV-2 protease cleavage of other substrate peptides with Ki values in the 5-20 microM range. Molecular modeling of the RSV protease with the p2-p10 peptide docked in the substrate binding pocket and analysis of a series of single-amino acid-substituted p2-p10 peptide analogues suggested that this peptide is inhibitory because of the potential of a serine residue in the P1' position to interact with one of the catalytic aspartic acid residues. To open the binding pocket and allow rotational freedom for the serine in P1', there is a further requirement for either a glycine or a polar residue in P2' and/or a large amino acid residue in P3'. The amino acid residues in P1-P4 provide interactions for tight binding of the peptide in the substrate binding pocket.     

Zinc inhibition of renin and the protease from human immunodeficiency virus type 1

We report here for the first time that Zn2+ is an effective inhibitor of renin and the protease from HIV-1, two aspartyl proteinases of considerable physiological importance. Inhibition of renin is noncompetitive and is accompanied by binding of 1 mol of Zn2+/mol of enzyme. Depending on the substrate, inhibition of the HIV protease by Zn2+ can be either competitive or noncompetitive, but in neither case is loss of activity due to disruption of the protease dimer. Inhibition of both enzymes is first order with respect to Zn2+ and is rapidly reversed by addition of EDTA. Ki values are strongly pH dependent and optimal in the range of 20 microM at or above pH 7. All of the data in hand suggest that the inhibitory effect of Zn2+ is a consequence of its binding at, or near, the active-site carboxyl groups of these aspartyl proteinases. This inhibition of the viral enzyme may help to explain some of the beneficial effects seen in AIDS patients who have received Zn2+ therapy.     

The inhibitory activity of a peptide derivative against the growth of simian immunodeficiency virus in C8166 cells.

The peptide derivative Ro 31-8959 is a potent and selective inhibitor of the aspartic proteinases encoded by HIV-1 and HIV-2 and it arrests the growth of both viruses in cell culture. We have demonstrated similar effects against the simian immunodeficiency virus SIVmac251 in the human T-cell line, C8166 (ED50 = 6nM) with a therapeutic index of 4,500. The antiviral activity of Ro 31-8959 was 250 and 22 times greater than that of ddI and ddC, respectively. The mode of action was confirmed by accumulation of the polyprotein p55 with concomitant reduction of the cleavage product, p27, and by the production of immature virions.     

Interdomain hydrolysis of a truncated Pseudomonas exotoxin by the human immunodeficiency virus-1 protease

The specificity of HIV-1 (human immunodeficiency virus-1) protease has been evaluated relative to its ability to cleave the three-domain Pseudomonas exotoxin (PE66) and related proteins in which the first domain has been deleted or replaced by a segment of CD4. Native PE66 is not hydrolyzed by the HIV-1 protease. However, removal of its first domain produces a molecule which is an excellent substrate for the enzyme. The major site of cleavage in this truncated exotoxin, called LysPE40, occurs in a segment that connects its two major domains, the translocation domain (II), and the ADP-ribosyltransferase (III). This interdomain region contains the sequence ...Asn-Tyr-Pro-Thr... which is similar to that surrounding the scissile Tyr-Pro bond in the gag precursor polyprotein, a natural substrate of the HIV-1 protease. Nevertheless, it is not this sequence that is recognized and cleaved by the enzyme, but one 6 residues away, ...Ala-Leu-Leu-Glu... in which the Leu-Leu peptide bond is hydrolyzed. A second, slower cleavage takes place at the Leu-Ala bond 3 residues in from the NH2 terminus of LysPE40. When domain I of PE66 is replaced by a segment comprising the first two domains of CD4, the resulting chimeric protein is hydrolyzed at the same Leu-Leu bond by HIV-1 protease. Enzyme activities toward synthetic peptides modeled after the sequences defined above in LysPE40 are in complete accord, relative to specificity, kinetics, and pH optimum, with results obtained in the hydrolysis of the parent protein. These findings demonstrate that ideas concerning the specificity of the HIV-1 protease that are based solely upon its processing of natural viral polyproteins can be expanded by evaluation of other multidomain proteins as substrates. Moreover, it would appear that it is not a particular conformation, but sequence and accessibility that play the dominant role in defining sites in a protein substrate that are susceptible to hydrolysis by the enzyme.     

Isolation and characterization of proteinases from the sarcocarp of snake-gourd fruit

Seven proteinases were isolated from the fruit of snake-gourd, Trichosanthes cucumeroides Maxim. Their isozymes are all serine proteinases, and homologous in their respective molecular weights, amino acid compositions, and enzymatic properties. Their molecular weight was estimated to be about 50,000. Using casein as a substrate, the maximum activity was found in the alkaline pH region. The optimum temperature using casein was 70 degrees C at pH 7.3. The enzymes were strongly inhibited by diisopropyl fluorophosphate and not inhibited by inhibitors of sulfhydryl or metalloproteases. The reduced and S-carboxymethylated insulin B-chain was used as a substrate in an investigation of the specificity. The enzyme was found to have a wide specificity for this substrate but preferentially hydrolyzed the peptide bonds involving the carboxyl groups of charged amino acid such as S-cm-cysteine, glutamic acid, histidine, arginine, and lysine. Experimental evidence indicated that the snake-gourd proteinases are similar in their properties to cucumisin, which is isolated from the sarcocarp of melon fruit.