Substrate-Dependent Mechanisms in the Catalysis of Human Immunodeficiency Virus Protease

Polgár, László; Szeltner, Zoltán; Boros, Imre

doi:10.1021/bi00197a040

Cited by 68 publications

(83 citation statements)

References 24 publications

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“…2-Aminobenzoyl-Thr-Ile-Nle-Phe(NO 2 )-Gln-Arg (substrate QR) and 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO 2 )-Glu-Arg (substrate ER) were prepared by solid phase synthesis (ABI 431A) and purified by high performance liquid chromatography. Lys-Ala-Arg-ValLeu-Phe(NO 2 )-Gln-Ala-Nle (substrate Q) and Lys-Ala-Arg-Val-LeuPhe(NO 2 )-Glu-Ala-Nle (substrate E) have already been described (3).…”

Section: Methodsmentioning

confidence: 99%

“…In a previous study (3), we have shown that a substrate of moderate specificity, Lys-Ala-Arg-Val-Leu-Phe(NO 2 )-Gln-AlaNle (substrate Q) is hydrolyzed at the Leu-Phe(NO 2 ) bond about 40 times faster by HIV-1 protease when Glu is substituted for the Gln in P2Ј. To establish the catalytic role of Glu in P2Ј, in this work the fluorogen 2-aminobenzoyl-Thr-Ile-NlePhe(NO 2 )-Gln-Arg (4) has been chosen which is one of the most effective substrates of HIV-1 protease.…”

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confidence: 88%

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Rate-determining Steps in HIV-1 Protease Catalysis

Szeltner

Polgár

1996

Journal of Biological Chemistry

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The human immunodeficiency virus type-1 (HIV-1) encodes a protease which is essential for the production of infectious virus. The protease prefers substrates that contain glutamic acid or glutamine at the P2 position. The catalytic role of these residues has been studied by using a highly specific fluorogen substrate, 2-aminobenzoyl-Thr-Ile-Nle-Phe(NO 2 )-Gln-Arg (substrate QR), and its counterpart (substrate ER) containing Glu in place of Gln. The newly designed substrate ER that contains a pair of charged residues at P2 and P3 sites is the most specific substrate described so far for HIV-1 protease. The specificity rate constant (k cat /K m ‫؍‬ 2.1 ؋ 10 7 M ؊1 s ؊1 ) approaches, but does not reach, the diffusion limit. This follows from the appreciable solvent kinetic deuterium isotope effects on the rate constants, indicating that, independent of the salt concentration, the rate-limiting step of the catalysis is a chemical process rather than a physical one. The reaction also has positive entropy of activation. On the other hand, the rate-limiting step for substrate QR changes with increasing salt concentration from a physical to chemical step, while the negative activation entropy becomes positive. The rate increase with substrate ER is 50-fold with respect to substrate QR in the presence of 0.1 M NaCl and diminishes to 3.5-fold at 2.0 M NaCl concentration, as a consequence of a considerable rate increase at high salt concentration with substrate QR but not with substrate ER. The K m value is much lower for the substrate ER (0.8 M) than for substrate QR (15 M), indicating a more effective binding for substrate ER at 0.1 M NaCl. Unexpectedly, the strong binding appears to be achieved by the unionized form of Glu in P2, as follows from the remarkably different pH-rate profiles for substrates QR and ER. The effective binding elicited by the glutamic acid may be utilized in designing inhibitors for therapeutic purposes.The HIV-1 1 protease is essential for the specific processing of large viral polyproteins into individual structural proteins and enzymes (1, 2). It exists as a homodimer of identical polypeptide chains, each consisting of 99 amino acid residues. A long cleft formed at the interface of the dimer harbors the catalytically competent aspartyl residues, Asp-25 and Asp-25Ј (the prime refers to the second subunit), contributed by each subunit. The cleft also contains eight subsites that accommodate P4, P3, P2, P1, P1Ј, P2Ј, P3Ј, and P4Ј residues of an octapeptide substrate. The substrate is cleaved between the P1 and P1Ј residues.In a previous study (3), we have shown that a substrate of moderate specificity, Lys-Ala-Arg-Val-Leu-Phe(NO 2 )-Gln-AlaNle (substrate Q) is hydrolyzed at the Leu-Phe(NO 2 ) bond about 40 times faster by HIV-1 protease when Glu is substituted for the Gln in P2Ј. To establish the catalytic role of Glu in P2Ј, in this work the fluorogen 2-aminobenzoyl-Thr-Ile-NlePhe(NO 2 )-Gln-Arg (4) has been chosen which is one of the most effective substrates of HIV-1 protease. Upon binding, a ...

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Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 88%

Rate-determining Steps in HIV-1 Protease Catalysis

Szeltner

Polgár

1996

Journal of Biological Chemistry

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“…The cycle initiates with water activation by hydrogen-bonding interactions with the active site aspartate residues (1) (6, 7). Bell-shaped pH-rate profiles (8)(9)(10)(11)(12) have revealed an acidic pH optimum, and structural studies (13,14) suggest that the reactant-bound enzyme contains a single protonated Asp (with a di-Asp, net charge of −1) facilitating a general acid-base mechanism, which is common among many Asp proteases (7,15). Nucleophilic attack by the water generates a reversible gem-diol intermediate, (3) which has been observed structurally (16)(17)(18) and identified experimentally (19,20).…”

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confidence: 99%

Transition states of native and drug-resistant HIV-1 protease are the same

Kipp¹,

Hirschi²,

Wakata³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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HIV-1 protease is an important target for the treatment of HIV/ AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary 14 at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.aspartyl protease | protease mechanism | transition-state structure | drug design

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“…However, we assumed that both retroviral proteases follow a similar catalytic reaction. Kinetic analysis has shown that HIV protease-catalyzed hydrolysis can have two different slow steps (Hyland et ai., 1991;Polgar et al, 1994). One of the slow steps was proposed to be the binding of Table 2 are shown.…”

Section: Interaction Energymentioning

confidence: 99%

“…Reaction coordinates consistent with experimental data for HIV protease catalysis from enzyme (E) and substrate ( S ) via intermediate (I) to enzyme and products (P) are shown. Two possible slow steps (Hyland et al, 1991;Polgar et al, 1994) Discussion of the entropic contribution…”

Section: E + P E + S 4 -W E Smentioning

confidence: 99%

Molecular mechanics calculations on rous sarcoma virus protease with peptide substrates

Weber

Harrison

1997

Protein Science

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Molecular models of Rous sarcoma virus (RSV) protease and 20 peptide substrates with single amino acid substitutions at positions from P4 to P3', where the scissile bond is between P1 and Pl', were built and compared with kinetic measurements. The unsubstituted peptide substrate, Pro-Ala-Val-Ser-Leu-Ala-Met-Thr, represents the NC-PR cleavage site of RSV protease. Models were built of two intermediates in the catalytic reaction, RSV protease with peptide substrate and with the tetrahedral intermediate. The energy minimization used an algorithm that increased the speed and eliminated a cutoff for nonbonded interactions. The calculated protease-substrate interaction energies showed correlation with the relative catalytic efficiency of peptide hydrolysis. The calculated interaction energies for the 8 RSV protease-substrate models with changes in P1 to P1' next to the scissile bond gave the highest correlation coefficient of 0.79 with the kinetic measurements, whereas all 20 substrates showed the lower, but still significant correlation of 0.46. Models of the tetrahedral reaction intermediates gave a correlation of 0.72 for the 8 substrates with changes next to the scissile bond, whereas a correlation coefficient of only 0.34 was observed for all 20 substrates. The differences between the energies calculated for the tetrahedral intermediate and the bound peptide gave the most significant correlation coefficients of 0.90 for models with changes in PI and Pl', and 0.56 for all substrates. These results are compared to those from similar calculations on HIV-1 protease and discussed in relation to the rate-limiting steps in the catalytic mechanism and the entropic contributions.Keywords: aspartic proteases; calculated interaction energies; RSV protease; substrate binding; transition state The Rous sarcoma virus (RSV) protease is a member of the aspartic protease family and is enzymatically active as a dimer. The crystal structure of RSV protease Jaskolski et al., 1991) has a three-dimensional fold similar to that of HIV-1 protease Weber, 1990). Although there are many crystal structures of HIV protease with different inhibitors (for review, see Wlodawer & Erickson, 1993), there is no crystal structure of RSV protease in the complex with an inhibitor. Instead, the structure of the RSV protease-substrate complex has been modeled by analogy to the structures of HIV protease with peptide-like inhibitors, as described in Grinde et al. (1992). RSV and HIV-1 proteases hydrolyze different cleavage sites in their viral polyprotein substrates (Oroszlan & Luftig, 1990), and RSV protease does not cleave peptides representing the natural substrates of HIV-I protease . The molecular basis of substrate specificity of RSV and HIV-1 proteases is not obvious, due to the lack of a clear consensus sequence in the different cleavage sites. The substrate specificity of RSV protease has been analyzed by kinetic measurements of hydrolysis of peptides that represent the natural cleavage sites, and variations of these peptides with dif...

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Substrate-Dependent Mechanisms in the Catalysis of Human Immunodeficiency Virus Protease

Cited by 68 publications

References 24 publications

Rate-determining Steps in HIV-1 Protease Catalysis

Rate-determining Steps in HIV-1 Protease Catalysis

Transition states of native and drug-resistant HIV-1 protease are the same

Molecular mechanics calculations on rous sarcoma virus protease with peptide substrates

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