Catalytic contribution of flap-substrate hydrogen bonds in "HIV-1 protease" explored by chemical synthesis.

Baca, Manuel; Kent, Stephen B. H.

doi:10.1073/pnas.90.24.11638

Cited by 68 publications

(49 citation statements)

References 30 publications

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“…To explore the role of flap backbone hydrogen bonds in catalysis, we synthesized both a monoester [Ile50, O-Ile50′]HIV-1 protease (enzyme 9) and the corresponding diester [O-Ile50,O-Ile50′] HIV-1 protease (enzyme 10), and obtained high-resolution X-ray structures for their complexes with substrate-based inhibitors [for catalytic activities see Table 1; values obtained were generally comparable with previous experimental data ( 24,25)]. Interestingly, in the X-ray structures of the ester analogue enzymes complexed with the reduced isostere MVT-101 inhibitor, electron density for the nonnucleophilic water molecule (water 301) bridging the flaps of the enzyme and the carbonyls of the inhibitor was found to be much less intense (Fig.…”

Section: Molecular Basis Of a Protein Dynamics Feedback Catalytic Mecsupporting

confidence: 54%

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis

Torbeev¹,

Raghuraman

Hamelberg

et al. 2011

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

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We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations. We show that conformational isomerization in the flaps is correlated with structural reorganization of residues in the active site, and that it is preorganization of the active site that is a rate-limiting factor in catalysis.enzyme catalysis | HIV protease | protein dynamics | protein NMR

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Section: Molecular Basis Of a Protein Dynamics Feedback Catalytic Mecsupporting

confidence: 54%

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis

Torbeev¹,

Raghuraman

Hamelberg

et al. 2011

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

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“…Although it was not possible to perfonn active site titrations for the mutant enzymes owing to their resistance against transition-state analogue inhibitors, the catalytic constants calculated from the protein content were dramatically reduced (Table 2). Similarly, a several thousandfold lower k,,, value was determined for a chemically engineered HIV-1 PR [25], and for certain active-site mutants of the tethered dimer [20]. In spite of the great loss, sufficient enzyme activity was measurable which allowed kinetic characterization of the mutants.…”

Section: Resultsmentioning

confidence: 99%

“…Rather, the flaps seem to be necessary for efficient catalytic activity of the protease. Recently, the role of the flap was studied using chemically synthesized HIV protease, in which the -CONH-group between Gly49 and lle50 was replaced with a -COS-bond [25]. Due to the proposed lack of ability to coordinate the unique water molecule at the tip of the flap, this backbone-modified enzyme showed a several-thousandfold reduction in catalytic activity (kcat) but normal substrate specificity and affinity (K,,,).…”

Section: Hydrolysis Of Oligopeptides Representing Other Naturally Occmentioning

confidence: 99%

Activity of Tethered Human Immunodeficiency Virus 1 Protease Containing Mutations in the Flap Region of One Subunit

Tőzsér

Yin

Cheng

et al. 1997

European Journal of Biochemistry

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USA 87, 9660-96641 and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Esclzerichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K,,, and decrease in k,,, values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P, and P; substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.Keywords: human immunodeficiency virus protease; flap mutants ; oligopeptide substrate; substrate specificity; enzyme kinetics.The protease (PR) of human retroviruses is an attractive target for chemotherapy of virus infection and associated diseases, including AIDS [I]. Knowledge of the specificity and mechanism of action of the retroviral PR have been useful in the design of potent inhibitors. The retroviral PR are aspartyl enzymes and are active as dimers of two identical subunits. Comparison of the crystal structures of human immunodeficiency virus 1 (HIV-1) PR with those of HIV-1 PR-inhibitor complexes (for review see [2]) suggested that during binding of the ligand, the two flaps close down on the ligand, providing a hydrophobic environment. The two flaps have an interchain hydrogen-bond connecting their tips. It is not clear whether the flaps contribute to the specificity and catalytic efficiency of the PR. In contrast to the flaps of retroviral PR, the cellular bilobal aspartic proteinases have only one flap, which cannot completely close down on the substrate, and there is no coordinated water molecule as observed in HIV PR-inhibitor structures [3-41.We have tested the effect of mutations in the flap region on the activity and specificity of HIV-1 PR. We used a single-chain tethered-dimer of HIV-1 PR [5]. The crystal structure of this tethered-dimer was recently determined [6] and is nearly identical to the structure of the wild-type two-chain protease. To explore the role of the hydrophobic nature of the amino acid residue at the tip of the flap (Ile50), we introduced a negatively charged (Asp) and a positively charged (His) residue into this position. We introduced mutations only into the flap region of one subunit. The mutation of only one flap was...

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“…1), with each subunit being composed of 99 amino acids [9][10][11]. As in all the other presently-known aspartyl proteases, the active site is located at the interface between the two subunits and features a triplet of conserved residues Asp, Thr, and Gly from each subunit [12].…”

Section: Introductionmentioning

confidence: 99%