Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.16 (HIV-1 protease)
 2,107 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RNase H domain of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was released from recombinant DHFR-RNase H fusion protein by the action of HIV-1 protease and crystallized as large trigonal prisms that diffract x-rays to at least 2.4-A resolution. The protease cleavage occurred 18 residues away from the Phe440-Tyr441 site reported to be processed during maturation of the reverse transcriptase heterodimer. Mutagenesis of the protease-sensitive region (residues 430-440), which is part of the crystallized domain, indicates that any alteration of the wild-type sequence results in increased proteolysis of the p66 subunit. A model of asymmetric processing in HIV-1 reserve transcriptase which involves partial unfolding of the RNase H domain is proposed based on these results and the recently reported three-dimensional structure of this domain.
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PMID:Proteolytic release and crystallization of the RNase H domain of human immunodeficiency virus type 1 reverse transcriptase. 171 88

An in vitro protein synthesizing system was modified to facilitate the improved, site-specific incorporation of unnatural amino acids into proteins via readthrough of mRNA nonsense (UAG) codons by chemically misacylated suppressor tRNAs. The modified system included an S-30 extract derived from Escherichia coli that expresses a temperature-sensitive variant of E. coli release factor 1 (RF1). Mild heat treatment of the S-30 extract partially deactivated RF1 and improved UAG codon readthrough by as much as 11-fold, as demonstrated by the incorporation of unnatural amino acids into positions 25 and 125 of HIV-1 protease and positions 10 and 22 of E. coli dihydrofolate reductase. The increases in yields were the greatest for those amino acids normally incorporated poorly in the in vitro protein synthesizing system, thus significantly enhancing the repertoire of modified amino acids that can be incorporated into the proteins of interest. The substantial increase in mutant protein yields over those obtained with an S-30 extract derived from an RF1 proficient E. coli strain is proposed to result from a relaxed stringency of termination by RF1 at the stop codon (UAG). When RF1 levels were depleted further, the intrinsic rate of DHFR synthesis increased, consistent with the possibility that RF1 competes not only at stop codons but also at other mRNA codons during peptide elongation. It thus seems possible that in addition to its currently accepted role as a protein factor involved in peptide termination, RF1 is also involved in functions that control the rate at which protein synthesis proceeds.
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PMID:Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids. 1039 57

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.
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PMID:Enzyme kinetics determined using calorimetry: a general assay for enzyme activity? 1155 13

The cleavage of a substrate protein by HIV-1 protease has been monitored in real time by the use of a dihydrofolate reductase fusion protein in which a fluorescence donor and a fluorescence acceptor were introduced into sites flanking the HIV-1 protease cleavage site. The amino acids 7-azatryptophan and dabcyl-1,2-diaminopropionic acid were introduced into specific sites of the DHFR fusion protein in an in vitro protein biosynthesizing system using two misacylated suppressor tRNAs, each of which recognized a specific, unique codon introduced into the mRNA. Excitation of the fluorescence acceptor in the initially expressed protein afforded no light production, consistent with quenching by fluorescence resonance energy transfer. Treatment of the elaborated protein with HIV-1 protease cleaved the protein between the fluorescence donor and acceptor, affording a time-dependent increase in fluorescence that was equal in magnitude to that produced by admixture of a stoichiometric amount of free 7-azatryptophan to the solution containing the intact protein.
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PMID:Fluorescence resonance energy transfer between unnatural amino acids in a structurally modified dihydrofolate reductase. 1217 3