Major enhancement of the affinity of an enzyme for a transition-state analog by a single hydroxyl group

The compound 1,6-dihydropurine ribonucleoside, prepared by reduction of nebularine in the presence of ultraviolet light, is bound by adenosine deaminase approximately 10(8)-fold less tightly than 6-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog. This difference in affinities, which is associated with the presence of a single hydroxyl group in the second compound, suggests the degree to which one or a few hydrogen bonds may stabilize the transition state in an enzyme reaction of this type.     

Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing

We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1.     

Three-dimensional solution structure of a single zinc finger DNA-binding domain

The three-dimensional solution structure of a zinc finger nucleic acid binding motif has been determined by nuclear magnetic resonance (NMR) spectroscopy. Spectra of a synthetic peptide corresponding to a single zinc finger from the Xenopus protein Xfin yielded distance and dihedral angle constraints that were used to generate structures from distance geometry and restrained molecular dynamics calculations. The zinc finger is an independently folded domain with a compact globular structure in which the zinc atom is bound by two cysteine and two histidine ligands. The polypeptide backbone fold consists of a well-defined helix, starting as alpha and ending as 3(10) helix, packed against two beta strands that are arranged in a hairpin structure. A high density of basic and polar amino acid side chains on the exposed face of the helix are probably involved in DNA binding.     

A fluorescent modification of adenosine triphosphate with activity in enzyme systems: 1,N 6 -ethenoadenosine triphosphate

A new, highly fluorescent adenosine triphophate (ATP) analog, 1,N(6) ethenoadenosine triphosphate, has been synthesized. Its fluorescence properties, including the long fluorescence lifetime and the possibility of detection at very low concentrations, in conjunction with its activity in the representative enzyme systems here reported, make it a valuable probe of enzymic mechanism and structure.     

Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade

The E6AP ubiquitin-protein ligase (E3) mediates the human papillomavirus-induced degradation of the p53 tumor suppressor in cervical cancer and is mutated in Angelman syndrome, a neurological disorder. The crystal structure of the catalytic hect domain of E6AP reveals a bilobal structure with a broad catalytic cleft at the junction of the two lobes. The cleft consists of conserved residues whose mutation interferes with ubiquitin-thioester bond formation and is the site of Angelman syndrome mutations. The crystal structure of the E6AP hect domain bound to the UbcH7 ubiquitin-conjugating enzyme (E2) reveals the determinants of E2-E3 specificity and provides insights into the transfer of ubiquitin from the E2 to the E3.     

Interference with feedback control; a mechanism of antimetabolite action

The action of an enzyme essential for tryptophan biosynthesis is inhibited by tryptophan and also by an analog of tryptophan. Similarly, histidine and one of its analogs inhibit the action of an enzyme essential for histidine biosynthesis. A mutant resistant to the histidine analog produces an apparently altered enzyme which is insensitive to both the analog and histidine.     

铜锌离子对超氧化物歧化酶构象及催化活性的影响

用ＣＤ光谱、紫外吸收光谱和荧光发射光谱监测了铜锌离子对超氧化物歧化酶构象的影响，结果表明脱铜锌后酶的二级结构有序度降低，三级结构发生去折叠，而且酶失活程度明显大于铜锌丢失程度。提示铜锌离子对于维持酶分子特定构象及对构象的稳定性具有重要作用。     

Redesigning trypsin: alteration of substrate specificity

A general method for modifying eukaryotic genes by site-specific mutagenesis and subsequent expression in mammalian cells was developed to study the relation between structure and function of the proteolytic enzyme trypsin. Glycine residues at positions 216 and 226 in the binding cavity of trypsin were replaced by alanine residues, resulting in three trypsin mutants. Computer graphic analysis suggested that these substitutions would differentially affect arginine and lysine substrate binding of the enzyme. Although the mutant enzymes were reduced in catalytic rate, they showed enhanced substrate specificity relative to the native enzyme. This increased specificity was achieved by the unexpected differential effects on the catalytic activity toward arginine and lysine substrates. Mutants containing alanine at position 226 exhibited an altered conformation that may be converted to a trypsin-like structure upon binding of a substrate analog.     

Structure of a peptide inhibitor bound to the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The structure of a 20-amino acid peptide inhibitor bound to the catalytic subunit of cyclic AMP-dependent protein kinase, and its interactions with the enzyme, are described. The x-ray crystal structure of the complex is the basis of the analysis. The peptide inhibitor, derived from a naturally occurring heat-stable protein kinase inhibitor, contains an amphipathic helix that is followed by a turn and an extended conformation. The extended region occupies the cleft between the two lobes of the enzyme and contains a five-residue consensus recognition sequence common to all substrates and peptide inhibitors of the catalytic subunit. The helical portion of the peptide binds to a hydrophobic groove and conveys high affinity binding. Loops from both domains converge at the active site and contribute to a network of conserved residues at the sites of magnesium adenosine triphosphate binding and catalysis. Amino acids associated with peptide recognition, nonconserved, extend over a large surface area.     

Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase

The crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase complexed with a 20-amino acid substrate analog inhibitor has been solved and partially refined at 2.7 A resolution to an R factor of 0.212. The magnesium adenosine triphosphate (MgATP) binding site was located by difference Fourier synthesis. The enzyme structure is bilobal with a deep cleft between the lobes. The cleft is filled by MgATP and a portion of the inhibitor peptide. The smaller lobe, consisting mostly of amino-terminal sequence, is associated with nucleotide binding, and its largely antiparallel beta sheet architecture constitutes an unusual nucleotide binding motif. The larger lobe is dominated by helical structure with a single beta sheet at the domain interface. This lobe is primarily involved in peptide binding and catalysis. Residues 40 through 280 constitute a conserved catalytic core that is shared by more than 100 protein kinases. Most of the invariant amino acids in this conserved catalytic core are clustered at the sites of nucleotide binding and catalysis.     

氮锌交互作用对黄棕壤锌形态的影响

选用南方牧场土壤，在盆栽条件下，研究了氮锌交互作用对土壤中锌形态和生物有效性的影响。结果表明：(1)随施氮[(NH4)2SO4]水平增加土壤pH显著降低。在不施锌或低量施氮条件下，施氮对速效锌影响不大；施锌条件下，高水平施氮显著提高了土壤速效锌含量。(2)随施锌水平增加，交换态锌、松结有机态锌、氧化锰结合态锌、无定形氧化铁结合态锌、紧结有机态锌所占比例明显增加，晶形氧化铁结合态锌及残留态锌所占比例明显下降：随施氮水平增加，土壤中交换态锌所占比例显著增加，松结有机态锌亦呈增加趋势。氮锌配施对土壤中交换态锌含量有协同效应，从而提高了锌对牧草的有效性。试验结果对南方牧场合理施肥具有指导意义。     

A dinuclear Ni(mu-H)Ru complex derived from H2

Models of the active site in [NiFe]hydrogenase enzymes have proven challenging to prepare. We isolated a paramagnetic dinuclear nickel-ruthenium complex with a bridging hydrido ligand from the heterolytic cleavage of H2 by a dinuclear NiRu aqua complex in water under ambient conditions (20 degrees C and 1 atmosphere pressure). The structure of the hexacoordinate Ni(mu-H)Ru complex was unequivocally determined by neutron diffraction analysis, and it comes closest to an effective analog for the core structure of the proposed active form of the enzyme.     

牛乳中超氧化物歧化酶(SOD)的提纯与鉴定

新鲜牛乳经超速离心,氯仿乙醇处理,丙酮沉淀、透析、ＤＥＡＥ纤维素柱层析等步骤使其中Ｃｕ－ＺｎＳＯＤ得到了纯化,提纯后酶的比活为１４９．５Ｖ／ｍｇ。该酶对Ｈ２Ｏ２敏感,并且在２７２ｎｍ和６７５ｎｍ处各有一吸收峰,证明被纯化的酶是Ｃｕ－ＺｎＳＯＤ。     

Molecular basis of proton motive force generation: structure of formate dehydrogenase-N

The structure of the membrane protein formate dehydrogenase-N (Fdn-N), a major component of Escherichia coli nitrate respiration, has been determined at 1.6 angstroms. The structure demonstrates 11 redox centers, including molybdopterin-guanine dinucleotides, five [4Fe-4S] clusters, two heme b groups, and a menaquinone analog. These redox centers are aligned in a single chain, which extends almost 90 angstroms through the enzyme. The menaquinone reduction site associated with a possible proton pathway was also characterized. This structure provides critical insights into the proton motive force generation by redox loop, a common mechanism among a wide range of respiratory enzymes.     

Structure of a site-2 protease family intramembrane metalloprotease

Regulated intramembrane proteolysis by members of the site-2 protease (S2P) family is an important signaling mechanism conserved from bacteria to humans. Here we report the crystal structure of the transmembrane core domain of an S2P metalloprotease from Methanocaldococcus jannaschii. The protease consists of six transmembrane segments, with the catalytic zinc atom coordinated by two histidine residues and one aspartate residue approximately 14 angstroms into the lipid membrane surface. The protease exhibits two distinct conformations in the crystals. In the closed conformation, the active site is surrounded by transmembrane helices and is impermeable to substrate peptide; water molecules gain access to zinc through a polar, central channel that opens to the cytosolic side. In the open conformation, transmembrane helices alpha1 and alpha6 separate from each other by 10 to 12 angstroms, exposing the active site to substrate entry. The structure reveals how zinc embedded in an integral membrane protein can catalyze peptide cleavage.     

Evaluation of intrinsic binding energy from a hydrogen bonding group in an enzyme inhibitor

This and two accompanying reports describe the intrinsic binding energy derived from a single hydrogen bond between an inhibitor and an enzyme. The results were obtained by comparing matched pairs of inhibitors of the zinc endopeptidase thermolysin that bind to the enzyme in an essentially identical manner but differ in the presence or absence of a specific hydrogen bond. This report describes five phosphorus-containing analogs of the peptides carbobenzoxy-Gly-Leu-X, in which the Gly-Leu peptide linkage is replaced with a phosphonate ester (-PO2(-)-O-). Values for the inhibition constants of these inhibitors show a direct relation with those of the corresponding phosphonamidate analogs (-PO2(-)-NH- in place of the Gly-Leu peptide moiety), which have been characterized previously as transition state analogs. However, each phosphonate ester is bound about 840 times more weakly than the analogous phosphonamidate, reflecting the loss of 4.0 +/- 0.1 kilocalories per mole in binding energy. From these results and the crystallographic analysis in the next report, it can be inferred that the value of 4.0 kilocalories per mole represents the intrinsic binding energy arising from a highly specific hydrogen binding interaction.     

Induced phenotypic resistance to an antimetabolite

Resistance to bacteriostasis by 2-thiazole alanine develops rapidly; however, such resistance is lost during growth in the absence of the analog. This induced resistance is accompanied by increased formation of an enzyme sensitive to 2-thiazole alanine. Maintenance of the elevated enzyme levels in growing cells, like resistance, requires the presence of the analog.     

Comment on "The mechanism for activation of GTP hydrolysis on the ribosome"

Voorhees et al. (Reports, 5 November 2010, p. 835) determined the structure of elongation factor Tu (EF-Tu) and aminoacyl-transfer RNA bound to the ribosome with a guanosine triphosphate (GTP) analog. However, their identification of histidine-84 of EF-Tu as deprotonating the catalytic water molecule is problematic in relation to their atomic structure; the terminal phosphate of GTP is more likely to be the proper proton acceptor.     

Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA

In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I.