Biomedical subjects

T W Ridky

Publications and source records attributed to T W Ridky.

7 recordsLinked to original sources

PubMed1998

Drug-resistant HIV-1 proteases identify enzyme residues important for substrate selection and catalytic rate.

A series of mutations, first identified in protease inhibitor-resistant HIV-1 viral isolates, were introduced into HIV-1 PR as individual substitutions. Mutants containing R8K, V32I, V82T, I84V, G48V/L90M, or V82T/I84V substitutions were analyzed for differences in substrate preference and catalytic efficiency using a set of single amino acid substituted HIV-1 CA-NCa cleavage site peptides. All mutants exhibited wild-type preference for large hydrophobic residues, especially Phe, in the P1' substrate position. Only the R8K and V32I mutants showed significant differences in subsite selection compared to wild-type enzyme. In a parallel study, the individual mutations R10K, L12V, I44V, A60M, I71V, and I108V were introduced into RSV PR. These amino acid positions are structurally equivalent to Arg8, Leu10, Val32, Met46, Ile54, and Ile84 in HIV-1 PR, respectively, which mutate in drug-resistance. The RSV R10K substitution significantly altered substrate specificity and catalytic rate, compared to wild-type, in a manner similar to that of the HIV-1 R8K mutant. Crystal structures of the RSV PR R10K, I44V, I71V, and Il08V mutant enzymes presented here indicate that each of these substitutions has little effect on the overall structure of the respective enzymes. Taken together, these data provide an explanation for the reported in vivo predilection for selection of large hydrophobic residues in the P1' substrate position of second locus mutations in the Gag polyprotein PR cleavage sites. The data also suggest that the selection of resistant enzymes is not simply limited to loss of binding to inhibitor but affects other steps in proteolysis.

Amino Acid Substitution↗

PubMed1998

Structural basis for specificity of retroviral proteases.

The Rous sarcoma virus (RSV) protease S9 variant has been engineered to exhibit high affinity for HIV-1 protease substrates and inhibitors in order to verify the residues deduced to be critical for the specificity differences. The variant has 9 substitutions (S38T, I42D, I44V, M73V, A100L, V104T, R105P, G106V, and S107N) of structurally equivalent residues from HIV-1 protease. Unlike the wild-type enzyme, RSV S9 protease hydrolyzes peptides representing the HIV-1 protease polyprotein cleavage sites. The crystal structure of RSV S9 protease with the inhibitor, Arg-Val-Leu-r-Phe-Glu-Ala-Nle-NH2, a reduced peptide analogue of the HIV-1 CA-p2 cleavage site, has been refined to an R factor of 0.175 at 2.4-A resolution. The structure shows flap residues that were not visible in the previous crystal structure of unliganded wild-type enzyme. Flap residues 64-76 are structurally similar to residues 47-59 of HIV-1 protease. However, residues 61-63 form unique loops at the base of the flaps. Mutational analysis indicates that these loop residues are essential for catalytic activity. Side chains of flap residues His 65 and Gln 63' make hydrogen bond interactions with the inhibitor P3 amide and P4' carbonyl oxygen, respectively. Other interactions of RSV S9 protease with the CA-p2 analogue are very similar to those observed in the crystal structure of HIV-1 protease with the same inhibitor. This is the first crystal structure of an avian retroviral protease in complex with an inhibitor, and it verifies our knowledge of the molecular basis for specificity differences between RSV and HIV-1 proteases.

Aspartic Acid Endopeptidases↗

PubMed1997

Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation.

Proteolytic processing of the Rous sarcoma virus (RSV) Gag precursor was altered in vivo through the introduction of amino acid substitutions into either the polyprotein cleavage junctions or the PR coding sequence. Single amino acid substitutions (V(P2)S and P(P4)G), which are predicted from in vitro peptide substrate cleavage data to decrease the rate of release of PR from the Gag polyprotein, were placed in the NC portion of the NC-PR junction. These substitutions do not affect the efficiency of release of virus-like particles from COS cells even though recovered particles contain significant amounts of uncleaved Pr76gag in addition to mature viral proteins. Single amino acid substitutions (A(P3)F and S(P1)Y), which increase the rate of PR release from Gag, also do not affect budding of virus-like particles from cells. Substitution of the inefficiently cleaved MA-p2 junction sequence in Gag by eight amino acids from the rapidly cleaved NC-PR sequence resulted in a significant increase in cleavage at the new MA-p2 junction, but again without an effect on budding. However, decreased budding was observed when the A(P3)F or S(P1)Y substitution was included in the NC-PR junction sequence between the MA and p2 proteins. A budding defect was also caused by substitution into Gag of a PR subunit containing three amino acid substitutions (R105P, G106V, and S107N) in the substrate binding pocket that increase the catalytic activity of PR. The defect appears to be the result of premature proteolytic processing that could be rescued by inactivating PR through substitution of a serine for the catalytic aspartic acid residue. This budding defect was also rescued by single amino acid substitutions in the NC-PR cleavage site which decrease the rate of release of PR from Gag. A similar budding defect was caused by replacing the Gag PR with two PR subunits covalently linked by four glycine residues. In contrast to the defect caused by the triply substituted PR, the budding defect observed with the linked PR dimer could not be rescued by NC-PR cleavage site mutations, suggesting that PR dimerization is a limiting step in the maturation process. Overall, these results are consistent with a model in which viral protein maturation occurs after PR subunits are released from the Gag polyprotein.

Animals↗

PubMed1996

Programming the Rous sarcoma virus protease to cleave new substrate sequences.

The Rous sarcoma virus protease displays a high degree of specificity and catalyzes the cleavage of only a limited number of amino acid sequences. This specificity is governed by interactions between side chains of eight substrate amino acids and eight corresponding subsite pockets within the homodimeric enzyme. We have examined these complex interactions in order to learn how to introduce changes into the retroviral protease (PR) that direct it to cleave substrates. Mutant enzymes with altered substrate specificity and wild-type or greater catalytic rates have been constructed previously by substituting single key amino acids in each of the eight enzyme subsites with those residues found in structurally related positions of human immunodeficiency virus (HIV)-1 PR. These individual amino acid substitutions have now been combined into one enzyme, resulting in a highly active mutant Rous sarcoma virus (RSV) protease that displays many characteristics associated with the HIV-1 enzyme. The hybrid protease is capable of catalyzing the cleavage of a set of HIV-1 viral polyprotein substrates that are not recognized by the wild-type RSV enzyme. Additionally, the modified PR is inhibited completely by the HIV-1 PR-specific inhibitor KNI-272 at concentrations where wild-type RSV PR is unaffected. These results indicate that the major determinants that dictate RSV and HIV-1 PR substrate specificity have been identified. Since the viral protease is a homodimer, the rational design of enzymes with altered specificity also requires a thorough understanding of the importance of enzyme symmetry in substrate selection. We demonstrate here that the enzyme homodimer acts symmetrically in substrate selection with each enzyme subunit being capable of recognizing both halves of a peptide substrate equally.

Aspartic Acid Endopeptidases↗

PubMed1996

Human immunodeficiency virus, type 1 protease substrate specificity is limited by interactions between substrate amino acids bound in adjacent enzyme subsites.

The specificity of the retroviral protease is determined by the ability of substrate amino acid side chains to bind into eight individual subsites within the enzyme. Although the subsites are able to act somewhat independently in selection of amino acid side chains that fit into each pocket, significant interactions exist between individual subsites that substantially limit the number of cleavable amino acid sequences. The substrate peptide binds within the enzyme in an extended anti-parallel beta sheet conformation with substrate amino acid side chains adjacent in the linear sequence extending in opposite directions in the enzyme-substrate complex. From this geometry, we have defined both cis and trans steric interactions, which have been characterized by a steady state kinetic analysis of human immunodeficiency virus, type-1 protease using a series of peptide substrates that are derivatives of the avian leukosis/sarcoma virus nucleocapsid-protease cleavage site. These peptides contain both single and double amino acid substitutions in seven positions of the minimum length substrate required by the retroviral protease for specific and efficient cleavage. Steady state kinetic data from the single amino acid substituted peptides were used to predict effects on protease-catalyzed cleavage of corresponding double substituted peptide substrates. The calculated Gibbs' free energy changes were compared with actual experimental values in order to determine how the fit of a substrate amino acid in one subsite influences the fit of amino acids in adjacent subsites. Analysis of these data shows that substrate specificity is limited by steric interactions between pairs of enzyme subsites. Moreover, certain enzyme subsites are relatively tolerant of substitutions in the substrate and exert little effect on adjacent subsites, whereas others are more restrictive and have marked influence on adjacent cis and trans subsites.

Amino Acid Sequence↗

PubMed1994

Mutational analysis of the substrate binding pockets of the Rous sarcoma virus and human immunodeficiency virus-1 proteases.

Mutations, designed by analysis of the crystal structures of Rous sarcoma virus (RSV) and human immunodeficiency virus type 1 (HIV-1) protease (PR), were introduced into the substrate binding pocket of RSV PR. The mutations substituted nonconserved residues of RSV PR, located within 10 A of the substrate, for those in structurally equivalent positions of HIV-1 PR. Changes in the activity of purified mutants were detected in vitro by following cleavage of synthetic peptides representing wild-type and modified RSV and HIV-1 gag and pol polyprotein cleavage sites. Substituting threonine for valine 104 (V104T), S107N, I44V, Q63M or deletion of residues 61-63 produced enzymes that were 2.5-7-fold more active than the wild type RSV PR. Substituting I42D, M73V, and A100L produced enzymes with lower activity, whereas a mutant that included both M73V and A100L was as active as wild type. Several substitutions altered the specificity for substrate. These include I42D and I44V, which contribute to the S2 and S2' subsites. These proteins exhibited HIV-1 PR specificity for P2- or P2'-modified peptide substrates but unchanged specificity with P4-, P3-, P1-, P1'-, and P3'-modified substrates. Changes in specificity in the S4 subsite were detected by deletion of residues 61-63. These results confirm the hypothesis that the subsites of the substrate binding pocket of the retroviral protease are capable of acting independently in the selection of substrate amino acids.

Amino Acid Sequence↗

PubMed1993

Molecular properties of pyruvate formate-lyase activating enzyme.

Pyruvate formate-lyase is a radical-containing enzyme that catalyzes the nonoxidative cleavage of pyruvate via a postulated homolytic mechanism. The formation of this enzymic radical in vitro requires an activating system composed of PFL-activating enzyme, S-adenosylmethionine, ferrous ion, a reduced flavin, DTT, and pyruvate as an allosteric effector. The need for large quantities of PFL-activating enzyme for biochemical and biophysical studies on the mechanism of protein radical formation has prompted us to clone the act gene and overexpress the gene product in Escherichia coli. Using PCR technology, the act gene was isolated and subcloned into various expression vectors. The overexpression of the protein was as high as 30-50% of the total cellular protein. However, the majority of the protein resided in the form of insoluble inclusion bodies. A procedure was developed to denature and isolate the inclusion bodies followed by refolding under anaerobic conditions. This purification method affords 5 mg of purified protein from 1 g of cells. Biochemical characterization demonstrated that the enzyme can bind one Fe(II) per protein monomer, and the protein did not exhibit any visible chromophore as previously observed. Co(II) and Cu(II) can be reconstituted into the protein with similar stoichiometries. Kinetic studies showed that the rate of radical formation was independent of ionic strength and the Km's for SAM and inactive PFL were determined to be 2.8 and 1.2 microM, respectively. Fluorescent binding data revealed that the Kd for SAM binding to the activating enzyme alone was comparable to the Km for SAM in the PFL activation indicating that the binding site for SAM resides on AE.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗