The hydrogen-atom transfer in soybean lipoxygenase-1 (SLO) exhibits a large kinetic isotope effect on k(cat) (KIE = 81) near room temperature and a very weak temperature dependence (E(act) = 2.1 kcal/mol). These properties are consistent with H small middle dot transfer that occurs entirely by a tunneling event. Mutants of SLO were prepared, and the temperature dependence of the KIE was measured, to test for alterations in the tunneling behavior. All mutants studied exhibit KIEs of similar, large magnitude at 30 degrees C, despite an up to 3 orders of magnitude change in k(cat). E(act) for two of the mutants (Leu(754) --> Ala, Leu(546) --> Ala) is larger than for wild-type (WT), and the KIE becomes slightly more temperature dependent. In contrast, Ile(553) --> Ala exhibits k(cat) and E(act) parameters similar to wild-type soybean lipoxygenase-1 (WT-SLO) for protiated substrate; however, the KIE is markedly temperature dependent. The behavior of the former two mutants could reflect increased reorganization energies (lambda), but the behavior of the latter mutant is inconsistent with this description. We have invoked a full H* tunneling model (Kuznetsov, A. M.; Ulstrup, J. Can. J. Chem. 1999, 77, 1085-1096) to explain the temperature dependence of the KIE, which is indicative of the extent to which distance sampling (gating) modulates hydrogen transfer. WT-SLO exhibits a very small E(act) and a nearly temperature-independent KIE, which was modeled as arising from a compressed hydrogen transfer distance with little modulation of the hydrogen transfer distance. The observations on the Leu(754) --> Ala and Leu(546) --> Ala mutants were modeled as arising from a slightly less compressed active site with greater modulation of the hydrogen transfer distance by environmental dynamics. Finally, the observed behavior of the Ile(553) --> Ala mutant indicates a relaxed active site with extensive involvement of gating to facilitate hydrogen transfer. We conclude that WT-SLO has an active site structure that is well organized to support hydrogen tunneling and that mutations perturb structural elements that support hydrogen tunneling. Modest alterations in active site residues increase lambda and/or increase the hydrogen transfer distance, thereby affecting the probability that tunneling can occur. These studies allow the detection and characterization of a protein-gating mode in catalysis.

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the alpha-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.

Controlling the biodistribution of nanoparticles upon intravenous injection is the key to achieving target specificity. One of the impediments in nanoparticle-based tumor targeting is the inability to limit the trafficking of nanoparticles to liver and other organs leading to smaller accumulated amounts in tumor tissues, particularly via passive targeting. Here we overcome both these challenges by designing nanoparticles that combine the specificity of antibodies with favorable particle biodistribution profiles, while not exceeding the threshold for renal filtration as a combined vehicle. To that end, ultrasmall silica nanoparticles are functionalized with anti-human epidermal growth factor receptor 2 (HER2) single-chain variable fragments to exhibit high tumor-targeting efficiency and efficient renal clearance. This ultrasmall targeted nanotheranostics/nanotherapeutic platform has broad utility, both for imaging a variety of tumor tissues by suitably adopting the targeting fragment and as a potentially useful drug delivery vehicle.

The effects of cotranslational protein modification on the process of protein folding are poorly understood. Time-resolved fluorescence energy transfer has been used to assess the impact of glycosylation on the conformational dynamics of flexible oligopeptides. The peptide sequences examined are selected from glycoproteins of known three-dimensional structure. The energy transfer modulation associated with N-linked glycosylation is consistent with the glycopeptides sampling different conformational profiles in water. Results show that glycosylation causes the modified peptides to adopt a different ensemble of conformations, and for some peptides this change may lead to conformations that are more compact and better approximate the conformation of these peptides in the final folded protein. This result further implies that cotranslational glycosylation can trigger the timely formation of structural nucleation elements and thus assist in the complex process of protein folding.The synthesis of many proteins takes place on membraneassociated ribosomes and follows the secretory pathway (1). These proteins are subject to a wide array of enzyme-catalyzed covalent chemical modifications. Early protein modification reactions, such as glycosylation, carboxylation, and hydroxylation, occur on protein substrates that are either only locally folded or in the form of "collapsed intermediates" (2). Because these protein processing events occur cotranslationally, questions arise as to the impact of each modification on the local peptide conformation and on the progress and efficiency of subsequent protein-folding events. Numerous observations attest to the importance of asparagine glycosylation for the appropriate folding and assembly of intact proteins. For example, the biosynthesis of proteins in the presence of N-linked glycosylation inhibitors, such as tunicamycin, often results in extensive aggregation associated with incomplete or incorrect folding (3-6).Previous studies on the conformational effects of protein glycosylation have principally relied upon NMR (7-9) and CD spectroscopy (10, 11). However, these methods present some limitations when considering the conformational dynamics of flexible peptides. Specifically, the time frame of NMR measurements is such that conformational averaging could obscure specific effects of glycosylation on a polypeptide framework. In addition, the molecular weights of the peptides and glycopeptides under evaluation are such that rotational correlation times would greatly affect the nuclear Overhauser effect (NOE) measurements, making the comparative studies difficult (12). Although CD spectroscopy operates on a faster time scale, the technique is mostly applicable to peptides with strong spectroscopic signals, such as a-helical motifs (10). In contrast, fluorescence energy transfer (FET) studies allow the assessment of specific conformational features, through the measurement of a single interprobe distance, on the same rapid time scale over which conformational fluctuations occu...

Bacterial resistance is eroding the clinical utility of existing antibiotics necessitating the discovery of new agents. Bacterial type II topoisomerase is a clinically validated, highly effective, and proven drug target. This target is amenable to inhibition by diverse classes of inhibitors with alternative and distinct binding sites to quinolone antibiotics, thus enabling the development of agents that lack cross-resistance to quinolones. Described here are novel bacterial topoisomerase inhibitors (NBTIs), which are a new class of gyrase and topo IV inhibitors and consist of three distinct structural moieties. The substitution of the linker moiety led to discovery of potent broad-spectrum NBTIs with reduced off-target activity (hERG IC 50 > 18 μM) and improved physical properties. AM8191 is bactericidal and selectively inhibits DNA synthesis and Staphylococcus aureus gyrase (IC 50 = 1.02 μM) and topo IV (IC 50 = 10.4 μM). AM8191 showed parenteral and oral efficacy (ED 50 ) at less than 2.5 mg/kg doses in a S. aureus murine infection model. A cocrystal structure of AM8191 bound to S. aureus DNA-gyrase showed binding interactions similar to that reported for GSK299423, displaying a key contact of Asp83 with the basic amine at position-7 of the linker.