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Nearly 100 years ago Michaelis and Menten published their now classic paper [Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung. Biochem. Z. 49, 333-369] in which they showed that the rate of an enzyme-catalyzed reaction is proportional to the concentration of the enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German yet is often quoted by English-speaking authors, we undertook a complete translation of the 1913 publication, which we provide as Supporting Information. Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our reanalysis of Michaelis and Menten's data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements but also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km × E0). [ABSTRACT FROM AUTHOR]

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3Dd2 (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation. [ABSTRACT FROM AUTHOR]

We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites. [ABSTRACT FROM AUTHOR]

A growing body of evidence suggests a connection between protein dynamics and enzymatic catalysis. In this paper, we present a variety of computational studies designed to investigate the role of protein dynamics in the detailed mechanism of peptidyl--prolyl cis--trans isomerization catalyzed by human cyclophilin A. The results identify a network of protein vibrations, extending from surface regions of the enzyme to the active site and coupled to substrate turnover. Indications are that this network may have a role in promoting catalysis. Crucial parts of this network are found to be conserved in 10 cyclophilin structures from six different species. Experimental evidence for the existence of this network comes from previous NMR relaxation studies, where motions in several residues, forming parts of this network, were detected only during substrate turnover. The high temperature factors (from X-ray crystal structures) associated with the network residues provide further evidence of these vibrations. Along with the knowledge of enzyme structure, this type of network could provide new insights into enzymatic catalysis and the effect of distant ligand binding on protein function. The procedure outlined in this paper is general and can be applied to other enzymatic systems as well. This presents an interesting opportunity; collaborative experimental and theoretical investigations designed to characterize in detail the nature and function of this type of network could enhance the understanding of protein dynamics in enzymatic catalysis. [ABSTRACT FROM AUTHOR]

Several laboratories have reported cooperative binding of S1 to actin in the presence of caldesmon. This cooperative binding has been interpreted with a model similar to that proposed for the binding of S1 to regulated actins in which the binding affinity of S1 is controlled by the position of the tropomyosin filaments. In a recent paper [Sen, A., Chen, Y., Yan, B., and Chalovich, J. M. (2001) Biochemistry 40, 5757-64], we showed qualitatively that S1 binding resulted in rapid dissociation of caldesmon from actin or actin-tropomyosin. This suggests that the cooperativity observed in the case of caldesmon is not due to a conformational change in actin-caldesmon but to the displacement of caldesmon. We show in this paper that the pure competitive binding model, in which both S1 and caldesmon are competing for the same binding sites on actin, can simulate quantitatively the effect of caldesmon on both the equilibrium and the kinetics of S1 binding to actin. This model successfully predicts an apparent cooperativity for the binding of S1 to actin-caldesmon without the need to assume multiple actin-caldesmon structures and produces a decreased rate of S1 binding to actin in the presence of caldesmon. This suggests that the inhibitory action of caldesmon on the actin-activated ATPase activity of myosin in solution and on the generation of active force in a contracting muscle may be simply due to the blocking of myosin binding sites on actin by caldesmon. [ABSTRACT FROM AUTHOR]

The chlorosome envelope of Chlorobaculum tepidum contains 10 polypeptides, three of which, CsmI, CsmJ, and CsmX, have an adrenodoxin-like domain harboring a single [2Fe-2S] cluster. Mutants that produced chlorosomes containing two, one, or none of these Fe–S proteins were constructed [Li, H., et al. (2013) Biochemistry 52, preceding paper in this issue (DOI: 10.1021/bi301454g)]. The electron paramagnetic resonance (EPR) spectra, g values, and line widths of the Fe–S clusters in individual CsmI, CsmJ, and CsmX proteins were obtained from studies with isolated chlorosomes. The Fe–S clusters in these proteins were characterized by EPR and could be differentiated on the basis of their g values and line widths. The EPR spectrum of wild-type chlorosomes could be simulated by a 1:1 admixture of the CsmI and CsmJ spectra. No contribution of CsmX to the EPR spectrum of chlorosomes was observed because of its low abundance. In chlorosomes that contained only CsmI or CsmJ, the midpoint potential of the [2Fe-2S] clusters was -205 or 8 mV, respectively; the midpoint potential of the [2Fe-2S] cluster in CsmX was estimated to be more oxidizing than -180 mV. In wild-type chlorosomes, the midpoint potentials of the [2Fe-2S] clusters were -348 mV for CsmI and 92 mV for CsmJ. The lower potential for CsmI in the presence of CsmJ, and the higher potential for CsmJ in the presence of CsmI, were attributed to interactions that occur when these proteins form complexes in the chlorosome envelope. The redox properties of CsmI and CsmJ are consistent with their proposed participation in the transfer of electrons to and from quenchers of energy transfer in chlorosomes. [ABSTRACT FROM AUTHOR]

GroEL, along with its coprotein GroES, is essential for ensuring the correct folding of unfolded or newly synthesized proteins in bacteria. GroEL is a complex, allosteric molecule, composed of two heptameric rings stacked back to back, that undergoes large structural changes during its reaction cycle. These structural changes are driven by the cooperative binding and subsequent hydrolysis of ATP, by GroEL. Despite numerous previous studies, the precise mechanisms of allosteric communication and the associated structural changes remain elusive. In this paper, we describe a series of all-atom, unbiased, molecular dynamics simulations over relatively long (50-100 ns) time scales of a single, isolated GroEL subunit and also a heptameric GroEL rin& in the presence and absence of ATP. Combined with results from a distance restraint-biased simulation of the single ring the atomistic details of the earliest stages of ATP-driven structural changes within this complex molecule are illuminated. Our results are in broad agreement with previous modeling studies of isolated subunits and with a coarse-grained, forcing simulation of the single sing. These are the first reported all-atom simulations of the GroEL single-ring complex and provide a unique insight into the role of charged residues K80, K277, R284, R285, and E388 at the subunit interface in transmission of the allosteric signal. These simulations also demonstrate the feasibility of performing all-atom simulations of very large systems on sufficiently long time scales on typical high performance computing facilities to show the origins of the earliest events in biologically relevant processes. [ABSTRACT FROM AUTHOR]

The Na+-stabilized alkali molten globule (B-state) of horse ferricytochrome c produced at pH 12.9 in 2 M NaCI has been studied to find out its relevance to the kinetic folding pathway of the protein. Details of stopped-flow kinetics indicate that the B-state when driven to fold at pH 11.5 goes through a submillisecond burst expansion to a state B' which must unfold to the base-denatured UB-state before folding to the alkaline native state, NB. This folding hallmark suggests that the B-state is a dead-end or off-pathway species, playing apparently an unclear role in the folding kinetics, interestingly, the folding kinetics of the B-state at a final pH of 7 is very similar to that observed for the guanidinium-unfolded ferricytochrome c (U) at pH 7. Both B and U exhibit a submillisecond burst phase followed by three observable phases, fast, medium, and slow, with matching rate constants for the fast phase, suggesting that B- and U-states share a common folding mechanism. Even the minima in the folding chevrons for the B- and U-states appear at the same denaturant concentration, but the former is shifted vertically upward and has shallower limbs, suggesting that the transition state relevant for the B-state folding is relatively more compact with greater surface burial allaying large-scale diffusive migration of chain segments. It is concluded that the B-state is not a good model for the kinetic molten globule of cytochrome c. Part of the reason for such atypical response of a typical molten globule may possibly be related to misligation of the ferric heme with lysyl side chains at the extreme alkaline pH required to produce the B-state. To eliminate this possibility, the companion paper [Bhuyan. A. K. (2010) Biochemistry (DOI 10.102l/bil100881n)] studies the B-state of ferrocytochrome c where ligation of the heme with any intrapolypeptide side chain is completely suppressed. The study concludes that the B-state is kinetically an abortive species. [ABSTRACT FROM AUTHOR]

Escherichia coli tyrosine kinase(Etk) regulates the export of pathogenic capsular polysaccharide (CPS) by intermolecularly autophosphorylating its C-terminal tyrosine cluster. The kinase Etk, however, needs to be first activated by the intramolecular phosphorylation of a tyrosine residue, Y574, next to the active site. The recently determined structure of Etk shows that dephosphorylated Y574 blocks the active site and prevents substrate access. After phosphorylation, the negatively charged P-Y574 side chain was previously postulated to flip out to associate with a positively charged R614, unblocking the active site. This proposed activation is unique among protein kinases; however, there is no direct structural evidence in support of this hypothesis. In this paper, we carried out molecular dynamics simulation, mutagenesis, and biochemical analysis to study the activation mechanism of Etk. Our simulation results are in excellent agreement with the proposed molecular switch involving P-Y574 and R614 in the activation of Etk. Further, we show that a previously unidentified residue, R572, modulates the rotation of the P-Y574 side chain through electrostatic interaction, slowing down the opening of the active site. Our enzymatic assays demonstrate that the R572A mutant of Etk possesses significantly increased kinase activity, providing direct experimental support for the unique activation mechanism of Etk. In addition, the simulation of the Etk Y574F mutant predicted short periods of unblocked active site by Y574F, in good agreement with the low kinase activity of this mutant. The C-terminal substrate peptide and the nucleotide cofactor were also docked into the active site, and their implications are discussed. [ABSTRACT FROM AUTHOR]

One of the major challenges in the postgenomic era is the functional assignment of proteins using sequence- and structure-based predictive methods coupled with experimental validation. We have used these approaches to investigate the structure and function of the Escherichia coli K-12 protein YfaU, annotated as a putative 4-hydroxy-2-ketoheptane-1,7-dioate aldolase (HpcH) in the sequence databases. HpcH is the final enzyme in the degradation pathway of the aromatic compound homoprotocatechuate. We have determined the crystal structure of apo-YfaU and the Mg2+--pyruvate product complex. Despite greater sequence and structural similarity to HpcH, genomic context suggests YfaU is instead a 2-keto-3-deoxy sugar aldolase like the homologous 2-dehydro-3-deoxygalactarate aldolase (DDGA). Enzyme kinetic measurements show activity with the probable physiological substrate 2-keto-3-deoxy-L-rhamnonate, supporting the functional assignment, as well as the structurally similar 2-keto-3-deoxy-L-mannonate and 2-keto-3-deoxy-L-lyxonate (see accompanying paper: Rakus, J. F., Fedorov, A. A., Fedorov, E. V., Glasner, M. E., Hubbard, B. K., Delli, J. D., Babbitt, P. C., Almo, S. C., and Gerlt, J. A. (2008) Biochemistry 47, 9944-9954). YfaU has similar activity toward the HpcH substrate 4-hydroxy-2-ketoheptane-1,7-dioate and synthetic substrates 4-hydroxy-2-ketopentanoic acid and 4-hydroxy-2-ketohexanoic acid. This indicates a relaxed substrate specificity that complicates the functional assignment of members of this enzyme superfamily. Crystal structures suggest these enzymes use an Asp-His intersubunit dyad to activate a metal-bound water or hydroxide for proton transfer during catalysis. [ABSTRACT FROM AUTHOR]

Human reticulocyte 15-lipoxygenase (15-hLO-1) and epithelial 15-lipoxygenase (15-hLO-2) have been implicated in a number of human diseases, with differences in their substrate specificity potentially playing a central role. In this paper, we present a novel method for accurately measuring the substrate specificity of the two 15-hLO isozymes and demonstrate that both cholate and specific LO products affect substrate specificity. The linoleic acid (LA) product, 13-hydroperoxyoctadienoic acid (13-HPODE), changes the (kcat/Km)AA/(kcat/Km)LA ratio more than 5-fold for 15-hLO-1 and 3-fold for 15-hLO-2, while the arachidonic acid (AA) product, 12-(S)-hydroperoxyeicosatetraenoic acid (12-HPETE), affects only the ratio of 15-hLO-1 (more than 5-fold). In addition, the reduced products, 12-(S)-hydroxyoctadecadienoic acid (13-HODE) and 12-(S)-hydroxyeicosatetraenoic acid (12-HETE), also affect substrate specificity, indicating that iron oxidation is not responsible for the change in the (kcat/Km)AA/(kcat/Km)LA ratio. These results, coupled with the dependence of the 1 5-hLO-1 kcat/Km kinetic isotope effect (Dkcat/Km) on the presence of 12-HPETE and 12-HETE, indicate that the allosteric site, previously identified in 15-hLO-1 [Mogul, R., Johansen, E., and Holman, T. R. (1999) Biochemistry 39, 4801-48071, is responsible for the change in substrate specificity. The ability of LO products to regulate substrate specificity may be relevant with respect to cancer progression and warrants further investigation into the role of this product-feedback loop in the cell. [ABSTRACT FROM AUTHOR]

The lipid peroxidation product 4-hydroxynonenal (4-HNE) is a signaling mediator with wide- ranging biological effects. In this paper, we report that disruption of mGsta4, a gene encoding the 4-HNE- conjugating enzyme mGSTA4-4, causes increased 4-HNE tissue levels and is accompanied by age-dependent development of obesity which precedes the onset of insulin resistance in 129/sv mice. In contrast, mGsta4 null animals in the C57BL/6 genetic background have normal 4-HNE levels and remain lean, indicating a role of 4-HNE in triggering or maintaining obesity. In mGsta4 null 129/sv mice, the expression of the acetyl-CoA carboxylase (ACC) transcript is enhanced several-fold with a concomitant increase in the tissue level of malonyl-CoA. Also, mitochondrial aconitase is partially inhibited, and tissue citrate levels are increased. Accumulation of citrate could lead to allosteric activation of ACC, further augmenting malonyl-CoA levels. Aconitase may be inhibited by 4-HNE or by peroxynitrite generated by macrophages which are enriched in white adipose tissue of middle-aged mGsta4 null 129/sv mice and, upon lipopolysaccharide stimulation, produce more reactive oxygen species and nitric oxide than macrophages from wild-type mice. Excessive malonyl-CoA synthesized by the more abundant and/or allosterically activated ACC in mGsta4 null mice leads to fat accumulation by the well-known mechanisms of promoting fatty acid synthesis and inhibiting fatty acid β-oxidation. Our findings complement the recent report that obesity causes both a loss of mGSTA4-4 and an increase in the level of 4-HNE [Grimsrud, P. A., et al. (2007) Mol. Cell. Proteomics 6, 624-637]. The two reciprocal processes are likely to establish a positive feedback loop that would promote and perpetuate the obese state. [ABSTRACT FROM AUTHOR]

This paper examines the functional mechanism of thioxolone, a compound recently identified as a weak inhibitor of human carbonic anhydrase II by Iyer et al. (2006) J. Biomol. Screening 11, 782-791. Thioxolone lacks sulfonamide, sulfamate, or hydroxamate functional groups that are typically found in therapeutic carbonic anhydrase (CA) inhibitors, such as acetazolamide. Analytical chemistry and biochemical methods were used to investigate the fate of thioxolone upon binding to CA II, including Michaelis-Menten kinetics of 4-nitrophenyl acetate esterase cleavage, liquid chromatography-mass spectrometry (LC-MS), oxygen-18 isotope exchange studies, and X-ray crystallography. Thioxolone is proposed to be a prodrug inhibitor that is cleaved via a CA II zinc-hydroxide mechanism known to catalyze the hydrolysis of esters. When thioxolone binds in the active site of CA II, it is cleaved and forms 4-mercaptobenzene-1,3-diol via the intermediate S-(2,4-thiophenyl)hydrogen thiocarbonate. The esterase cleavage product binds to the zinc active site via the thiol group and is therefore the active CA inhibitor, while the intermediate is located at the rim of the active-site cavity. The time-dependence of this inhibition reaction was investigated in detail. Because this type of prodrug inhibitor mechanism depends on cleavage of ester bonds, this class of inhibitors may have advantages over sulfonamides in determining isozyme specificity. A preliminary structure-activity relationship study with a series of structural analogues of thioxolone yielded similar estimates of inhibition constants for most compounds, although two compounds with bromine groups at the C1 carbon of thioxolone were not inhibitory, suggesting a possible steric effect. [ABSTRACT FROM AUTHOR]

Sensitivity amplification in signal transduction modules regulated by phosphorylation-dephosphorylation cycles and GTPases results from a new type of cooperativity which is fundamentally different from that of allosterism. This type of cooperativity, termed temporal cooperativity [Qian, H. (2003) Biophys. Chem. 105, 585–593], is analyzed in this paper through stochastic models for molecular interactions. Mathematical analysis is developed through a series of models with different levels of complexity, from which a simple conceptual model based on linear cooperativity is derived. The following are shown: (i) When both kinase and phosphatase are nonsaturating, the distribution of the number of activated substrate molecules is binomial. With increasing kinase activity, the peak of the distribution continuously moves toward 100% activation. (ii) When the kinase is saturated, i.e., zeroth order, the distribution is Poisson. (iii) When both enzymes are saturated, the distribution is geometric. Ultrasensitivity corresponds to an abrupt switching of the peak position of the distribution from 0 to 100%. The theory is applicable to a wide range of processes in cell signaling including the specificity and sensitivity of T-cell activation. [ABSTRACT FROM AUTHOR]

In this paper, a numerically detailed thermodynamic investigation of nucleosomal core particles is presented. The nonlinear Poisson-Boltzmann equation governs the electrostatic properties of both the DNA and histone protein. Brownian dynamics is used as the leading method, in combination with the analysis of the electrical features of the nucleosome. At elevated temperature, the structure of the nucleosome is destabilized by the decrease in electrical interactions of DNA-histone complexes, which can be explained with the EDL theory. Two obvious unwrapping transitions can be found, occurring within the temperature ranges 43-52 and 65-80 °C. The first transition is characterized by the melting of DNA terminal domains, and the feature of the second transition is the massive unwrapping of the DNA middle domain. It can be concluded that the nucleosomal DNA consists of two distinct structures, where the DNA terminal domains are less tightly bound to the histone than the DNA middle domain. [ABSTRACT FROM AUTHOR]

β-Lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by β-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by β-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of β-lactams (penicillins and cephalosporins) containing peptidoglycanmimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 Do-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor β-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and β-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design. [ABSTRACT FROM AUTHOR]

Many fungal rhodopsins, eukaryotic structural homologues of the archaeal light-driven proton pump bacteriorhodopsin, have been discovered in the course of genome sequencing projects. Recently, two fungal rhodopsins were characterized in vitro and exhibited very different photochemical behavior. Neurospora rhodopsin possesses a slow photocycle and shows no ion transport, reminiscent of sensory rhodopsins, while Leptosphaeria rhodopsin has a fast bacteriorhodopsin-like photocycle and pumps protons light-dependently. Such a dramatic difference is surprising considering the very high degree of sequence homology of the two proteins. In this paper, we investigate whether the chemical structure of a cytoplasmic carboxylic acid, the homologue of Asp-96 of bacteriorhodopsin serving as a proton donor for the retinal Schiff base, can define the photochemical properties of fungal rhodopsins. We studied mutants of Leptosphaeria rhodopsin in which this aspartic acid was replaced with Glu or Ash using spectroscopy in the infrared and visible ranges. We show that Glu at this position is inefficient as a proton donor similar to a nonprotonatable Asn. Moreover, this replacement induces long-range structural perturbations of the retinal environment, as evidenced by changes in the vibrational bands of retinal (especially, hydrogen-out-of-plane modes) and neighboring aspartic acids and water molecules. The conformational coupling of the mutation site to the retinal may be mediated by helical rearrangements as suggested by the changes in amide and proline vibrational bands. We conclude that the difference in the photochemical behavior of fungal rhodopsins from Leptosphaeria and Neurospora may be ascribed, to some extent, to the replacement of the cytoplasmic proton donor Asp with Glu. [ABSTRACT FROM AUTHOR]

β-Lactam antibiotics restrict bacterial growth by inhibiting DD-peptidases. These enzymes catalyze the final transpeptidation step in bacterial cell wall biosynthesis. Although much structural information is now available for these enzymes, the mechanism of the actual transpeptidation reaction has not been studied in detail. The reaction is known to involve a double-displacement mechanism with an acyl–enzyme intermediate, which can be attacked by water, specific amino acids, peptides, and other acyl acceptors. We describe in this paper an investigation of acyl acceptor specificity and assess the need for general base catalysis in the deacylation transition state of the Streptomyces R61 DD-peptidase. We show, by the criterion of solvent deuterium kinetic isotope effect measurements and proton inventories, that the transition states of specific and nonspecific substrates are very similar, at least with respect to proton motion. The transition states for attack (tetrahedral intermediate formation) by D-amino acids and Gly-L-Xaa dipeptides do not include a general base catalyst, while such catalysis is essential for reaction with water and D-α-hydroxy acids. D-α-Hydroxy acids act as acyl acceptors for glycyl substrates but not for more specific D-alanyl substrates; hydroxy acids actually behave, more generally, as mixed inhibitors of the DD-peptidase. The structural and mechanistic bases of these observations are discussed; they should inform transition state analogue design. [ABSTRACT FROM AUTHOR]

The scaffold of serine protease inhibitors plays a significant role in the process of religation which resists proteolysis of the inhibitor in comparison to a substrate. Although the role of the conserved scaffolding Asn residue was previously implicated in the maintenance of the binding loop conformation of Kunitz (STI) inhibitors, its possible involvement in the prevention of proteolysis is still unexplored. In this paper, we have investigated the specific role of the spacer Asn in the prevention of proteolysis through structural and biochemical studies on the mutants where Asn14 of winged bean chymotrypsin inhibitor (WCI) has been replaced by Gly, Ala, Thr, Leu, and Gin. A residue having no side chain or β-branching at the 14th position creates deformation and insufficient protrusion of the binding loop, and as a result N14G and N14T lose the ability to recognize proteases. Although the reactive site loop conformation of N14A and N14Q are almost identical to WCI, biochemical results present N14A as a substrate indicating that the methyl group of Ala14 is not suitable to capture the cleaved parts together for religation. The poor inhibitory power of N14L points toward the chemical incompatibility of Leu at the 14th position, although its size is the same as Asn; on the other hand, slight loss of inhibitory potency of N14Q is attributed to the inappropriate placement of the Gln14 polar head, caused by the strained accommodation of its bigger side chain. These observations collectively allow us to conclude that the side chain of spacer Asn fits snugly into the concave space of the reactive site loop cavity and its ND2 atom forms hydrogen bonds with the P2 and P1‰ carbonyl 0 at either side of the scissile bond holding the cleaved products together for religation. Through database analysis, we have identified such spacer asparagines in five other families of serine protease inhibitors with a similar disposition of their ND2 atoms, which supports our proposition. [ABSTRACT FROM AUTHOR]

Onconase (ONC) from Rana pipiens is the smallest member of the ribonuclease A (RNase A) superfamily. Despite a tertiary structure similar to RNase A, ONC is distinguished by an extremely high thermodynamic stability. In the present paper we have probed the significance of three structural regions, which exhibit structural peculiarities in comparison to RNase A, for the stability of ONC to temperature and guanidine hydrochloride induced denaturation: (i) the N-terminal pyroglutamate residue, (ii) the hydrophobic cluster between helix I and the first β-sheet, and (iii) the C-terminal disulfide bond. For this purpose, the enzyme variants

The ability to rationally engineer a protein with altered stability depends upon the detailed understanding of the role of noncovalent interactions in defining thermodynamic properties of proteins. In this paper, we used T. celer L30e as a model to address the question of the role of charge—charge interactions in defining the stability of this protein. A total of 26 single-site charge-to-alanine variants of this protein were generated, and the stability of these proteins was determined using thermal- and denaturant-induced unfolding. It was found that, although L30e is isolated from a thermophilic organism and is highly thermostable, some of the substitutions lead to a further increase in the transition temperature. Analysis of the effects of high ionic strength on the stabilities of L30e variants shows that the long-range charge—charge interactions are as important as the short-range (salt bridge) interactions. The changes in stabilities of the T. celer L30e protein variants were compared with the changes in the energy of charge—charge interactions calculated using different computational models. It was found that there is a good qualitative agreement between experimental and calculated data: for 70-80% (19-21 of 26, confidence p < 0.003) of the variants, computational models predict correctly the sign of the stability changes. In particular, computational models identify correctly those charged amino acid residue substitutions of which led to enhancement in thermostability. Thus, optimization of the charge—charge interactions might be a useful approach for the rational increase in protein stability. [ABSTRACT FROM AUTHOR]

Investigators have studied how proteins enforce nonstandard geometries on metal centers to assess the question of how protein structures can define the coordination geometry and binding affinity of an active-site metal cofactor. We have shown that cysteine-substituted versions of the TRI peptide series [AcG-(LKALEEK)4G-NH2] bind HgII and CdII in geometries that are different from what is normally found with thiol ligands in aqueous solution. A fundamental question has been whether this structural perturbation is due to protein influence or a change in the metal geometry preference. To address this question, we have completed linear free-energy analyses that correlate the association of three-stranded coiled coils in the absence of a metal with the binding affinity of the peptides to the heavy metals, HgII and CdII. In this paper, six new members of this family have been synthesized, replacing core leucine residues with smaller and less hydrophobic residues, consequently leading to varying degrees of self-association affinities. At the same time, studies with some smaller and longer sequenced peptides have also been examined. All of these peptides are seen to sequester HgII and CdII in an uncommon trigonal environment. For both metals, the binding is strong with micromolar dissociation constants. For binding of HgII to the peptides, the dissociation constants range from 2.4 × 10-5 M for Baby L12C to 2.5 × 10-9 M for Grand L9C for binding of the third thiolate to a linear HgII(pep)2 species. The binding of HgII to the peptide Grand L9C is similar in energetics for metal binding in the metalloregulatory protein, mercury responsive (merR), displaying ∼50% trigonal HgII formation at nanomolar metal concentrations. Approximately, 11 kcal/mol of the HgII(Grand L9C)ç stability is due to peptide interactions, whereas only 1—4 kcallmol stabilization results from HgII(RS)2 binding the third thiolate ligand. This further validates the hypothesis that the favorable tertiary interactions in protein systems such as merR go a long way in stabilizing nonnatural coordination environments in biological systems. Similarly, for the binding of CdII to the TM family, the dissociation constants range from 1.3 × 10-6 M for Baby L9C to 8.3 × 10-9 M for TM L9C, showing a similar nature of stable aggregate formation. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO) converts the energy from redox and oxygen chemistry to support proton translocation and create a transmembrane ΔμH+ used for ATP production. Molecular dynamics (MD) simulations were carried out to probe for the formation water chains capable of participating in proton translocation. Attention was focused on the region between and above the a and a3 hemes where well-defined water chains have not been identified in crystallographic studies. An arginine (R481) (Rhodobacter sphaeroides numbering), positioned between the D-propionates of the hemes, had been mutated in vivo to lysine and showed to have altered activity consistent with an altered proton conductance [Qian, J., Mills, D. A., Geren, L., Wang, K. F., Hoganson, C. W., Schmidt, B., Hiser, C., Babcock, G. 1., Durham, B., Millett, F., and Ferguson-Miller, S. (2004) Role of the conserved arginine pair in proton and electron transfer in cytochrome c oxidase, Biochemistry 43, 5748–5756; also see the accompanying paper by Mills et al.]. This mutant was created in silico, and the MD results for the mutant and wild type were compared to explore the effects on the formation of hydrogen-bonded water chains by this mutation. The simulations reveal the presence of hydrogen-bonded water chains that lead from E286 through the region above the hemes to the Mg2+, and from E286 to the heme a3 D-propionate and the binuclear center. The R481 K mutant does not form as many, or as extensive, water chains as wild-type CcO, due to a new conformation of residues in a large loop between helices Ill and IV in subunit I, indicating a reduction in the level of water chain formation in the mutant. This loop appears to play a role in controlling the formation of hydrogen-bonded water chains above the hemes. The results suggest a possible gating mechanism for proton movement that includes key residues W172 and Y175 on the loop and F282 on helix VI. [ABSTRACT FROM AUTHOR]

In parts 1 and 2 of this series [Hanson, U. T., McAnaney, T. B., Park, E. S., Rendell, M. B. P., Yarbrough, D. K., Chu, S. Y., Xi, L. X., Boxer, S. U., Montrose, M. H., and Remington, S. J. (2002) Biochemistry 41, 15477-15488; McAnaney, T. B., Park, E. S., Hanson, U. T., Remington, S. J., and Boxer, S. U. (2002) Biochemistry 41, 15489-15494], we described the structure, excited-state dynamics, and applications of pH-sensitive, ratiometric dual emission green fluorescent protein (deUFP) variants with fluorescence emission that is modulated between blue (λmax ≅ 465 nm) and green (λmax ≅ 515 nm) depending on the pH of the bulk solvent. In this paper, we consider the energetic origin of the dual emission properties of these GFP variants by examining the temperature dependence of the steady-state absorption and fluorescence emission. In most cases, the quantum yield of the green emission decreased as the temperature was lowered, indicating that the excited-state proton transfer (ES PT) which produces the green emitting form is an activated process. The activation energies of ESPT, determined by modeling the quantum yields of both blue and green emissions between 260 and 298 K in the context of a simple photocycle, were found to be larger at low pH than at high pH. These results indicate that the ratiometric dual emission properties of deGFP mutants are due to this pH-sensitive ESPT rate, combined with a modulation of the ground-state neutral and anionic chromophore populations with pH. The time-resolved fluorescence of one of the deGFP mutants was studied in detail. The time-resolved emission spectra of this mutant are the first ultrafast spectra obtained for a GFP. These spectra demonstrate that the rising kinetics for green emission, considered a hallmark of ESPT, is the sum of the contribution from both the neutral and intermediate anionic forms of the chromophore at the probe wavelength and may not be observed in all mutants that undergo ESPT, depending on the relative contributions of the two forms. [ABSTRACT FROM AUTHOR]

The misfolding of proteins into β-sheets and the subsequent aggregation of these sheets into fibrous networks underlies many diseases. In this paper, the role of peptide structure in determining the ordering of β-sheet aggregates and the morphology of fibrils and protofibrils is dissected. Using a series of peptides based on residues 109-122 of the Syrian hamster prion protein (H1) with a range of substitutions at position 117, the link between side chain interactions and β-sheet thermal stability has been investigated. The thermal stability of β-sheets is associated with the peptides' ability to adopt the same alignment as wild-type H1, with residue 117 in register across all β-strands [Silva, R. A. G. D., Barber-Armstrong, W., and Decatur, S. M. (2003) J. Am. Chem. Soc. 125, 13674-13675]. These aligned strands are capable of forming long, rigid, and twisted fibrils (as visualized by atomic force microscopy) which are thermostable. Peptides which do not adopt this strand alignment aggregate to form thin, flexible, and smooth protofibrils. The ability to form ordered aggregates, and thus to form twisted fibrils, is modulated by the structure of the side chain of residue 117. [ABSTRACT FROM AUTHOR]

FCP1, a phosphatase specific for the carboxyl-terminal domain of the largest subunit of RNA polymerase II, is regulated by the HJV-1 Tat protein, CK2, TFIJB, and the large subunit of TFIIF (RAP74). We have characterized the interactions of Tat and RAP74 with the BRCT-containing central domain of FCP1 (FCP1562-738). We demonstrated that FCP1 is required for Tat-mediated transactivation in vitro and that amino acids 562-685 of FCPI are necessary for Tat interaction in yeast two-hybrid studies. From sequence alignments, we identified a conserved acidic/hydrophobic region in FCP1 adjacent to its highly conserved BRCT domain. In vitro binding studies with purified proteins indicate that HIV-1 Tat interacts with both the acidic/hydrophobic region and the BRCT domain of FCP1, whereas RAP74436-517 interacts solely with a portion of the acidic/hydrophobic region containing a conserved LXXLL-like motif HIV-1 Tat inhibits the binding of RAP74436-517 to FCP1. In a companion paper (K. Abbott et al. (2005) Enhanced Binding of RNAPII CTD Phosphatase FCP1 to RAP74 Following CK2 Phosphorylation, Biochemistry 44, 2732-2745, we identified a novel CK2 site adjacent to this conserved LXXLL-like motif. Phosphorylation of FCP1562-619 by CK2 at this site increases binding to RAP74436-517, but this phosphorylation is inhibited by Tat. Our results provide insights into the mechanisms by which Tat inhibits the FCP1 CTD phosphatase activity and by which FCP1 mediates transcriptional activation by Tat. In addition to increasing our understanding of the role of HIV-1 Tat in transcriptional regulation, this study defines a clear role for regions adjacent to the BRCT domain in promoting important protein-protein interactions. [ABSTRACT FROM AUTHOR]

Phosphopantothenoylcysteine decarboxylase (PPC-DC) catalyzes the decarboxylation of the cysteine moiety of 4'-phosphopantothenoylcysteine (PPC) to form 4'-phosphopantetheine (PPantSH); this reaction forms part of the biosynthesis of coenzyme A. The enzyme is a member of the larger family of cysteine decarboxylases including the lantibiotic-biosynthesizing enzymes EpiD and MrsD, all of which use a tightly bound flavin cofactor to oxidize the thiol moiety of the substrate to a thioaldehyde. The thioaldehyde serves to delocalize the charge that develops in the subsequent decarboxylation reaction. In the case of PPC-DC enzymes the resulting enethiol is reduced to a thiol giving net decarboxylation of cysteine, while in EpiD and MrsD it is released as the final product of the reaction. In this paper, we describe the characterization of the novel cyclopropyl-substituted product analogue 4'-phospho-N-(1-mercaptomethyl-cyclopropyl)-pantothenamide (PPanASH) as a mechanism-based inhibitor of the human PPC-DC enzyme. This inhibitor alkylates the enzyme on Cys173, resulting in the trapping of a covalently bound enethiolate intermediate. When Cys173 is exchanged for the weaker acid serine by site-directed mutagenesis the enethiolate reaction intermediate also accumulates. This suggests that Cys173 serves as an active site acid in the protonation of the enethiolate intermediate in PPC-DC enzymes. We propose that this protonation step is the key mechanistic difference between the oxidative decarboxylases EpiD and MrsD (which have either serine or threonine at the corresponding position in their active sites) and PPC-DC enzymes, which also reduce the intermediate in an overall simple decarboxylation reaction. [ABSTRACT FROM AUTHOR]

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn2+ and Co2+ preferentially activate family II PPases; Mg2+ partially activates; and Zn2+ can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn2+ at 1.3 Å resolution, and, at 2.05 Å resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn2+. Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn2+ or Co2+, as an activator rather than Mg2+. Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn2+ and Co2+, which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn2+ activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205 - Gln81 - Gln80 form a hydrophilic channel to speed product release from the active site. [ABSTRACT FROM AUTHOR]

The enzymes involved in the biosynthesis of peptidoglycan are targets for the development of new antibiotics. The bifunctional high molecular weight (HMW) penicillin-binding proteins (PBPs), which contain both glycosyltransferase (GTase) and transpeptidase (TPase) activities, are particularly attractive targets because of their extracellular location. However, there is limited mechanistic or structural information about the GTase modules of these enzymes. In this paper, we describe the overexpression and characterization of the GTase module of Escherichia coli PBP1b, a paradigm of the HMW PBPs. We define the C-terminal boundary of the GTase module and show that the isolated module can be overexpressed at significantly higher levels than the full-length protein. The catalytic efficiency and other characteristics of the isolated module are comparable in most respects to the full-length enzyme. This work lays the groundwork for mechanistic and structural analysis of GTase modules. [ABSTRACT FROM AUTHOR]

Thermobifida fusca Cel9A-90, an unusual family 9 enzyme, is a processive endoglucanase containing a catalytic domain closely linked to a family 3c cellulose binding domain (Cel9A-68) followed by a fibronectin III-like domain and a family 2 cellulose binding domain. To study its catalytic mechanism, 12 mutant genes with changes in five conserved residues of Cel9A-68 were constructed, cloned, and expressed in Escherichia coil. The purified mutant enzymes were assayed for their activities on (carboxymethyl)cellulose, phosphoric acid-swollen cellulose, bacterial microcrystalline cellulose, and 2,4-dinitrophenyl β-D-cellobioside. They were also tested for ligand binding, enzyme processivity, and thermostability. The results clearly show that E424 functions as the catalytic acid, D55 and D58 are both required for catalytic base activity, and Y206 plays an important role in binding, catalysis, and processivity, while Y318 plays an important role in binding of crystalline cellulose substrates and is required for processivity. Several amino acids located in a loop at the end of the catalytic cleft (T245-L251) were deleted from Cel9A-68, and this enzyme showed slightly improved filter paper activity and binding to BMCC but otherwise behaved like the wild-type enzyme. The FnIII-like domain was deleted from Cel9A- 90, reducing BMCC activity to 43% of the wild type. [ABSTRACT FROM AUTHOR]

The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A2 (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow "activation" of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady- state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed. [ABSTRACT FROM AUTHOR]

Active transport of acetylcholine (ACh) by vesicular ACh transporter (YAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [³H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat YAChT expressed in PC12A1237 cells. Equilibrium titrations of [³H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the YAChT, NaCl did not mimic ACh chloride, and bafilomycin A1 and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the YAChT transport cycle. [ABSTRACT FROM AUTHOR]

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca2+/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of ∼ 10-20-10-18 moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor. [ABSTRACT FROM AUTHOR]

This paper describes the design of the highest affinity ligands for Grb2 SH3 domains reported so far. These compounds were designed by combining N-alkyl amino acid incorporation in a proline-rich sequence with subsequent dimerization of the peptoid sequence based on structural data and molecular modeling. Optimization of the linker size is discussed, and the N-alkyl amino acid incorporation into both monomeric halves is reported. Because the affinity for Grb2 of the optimized compounds was too high to be measured using the fluorescent modifications that they induce on the Grb2 emission spectrum, a competition assay was developed. In this test, Grb2 is pulled down from a cellular extract by the initial VPPPVPPRRR peptide bound to Sepharose beads. In the presence of competitors, the test quantifies the amount of Grb2 displaced from the beads. It has enabled us to determine a Ki value in the 10-10 M range for the highest affinity Grb2 peptoid analogue dimer. [ABSTRACT FROM AUTHOR]

Acetylcholine-receptor channels (AChRs) are allosteric membrane proteins that mediate synaptic transmission by alternatively opening and closing ("gating") a cation-selective transmembrane pore. Although ligand binding is not required for the channel to open, the binding of agonists (for example, acetylcholine) increases the closed ⇋ open equilibrium constant because the ion-impermeable -→ ion-permeable transition of the ion pathway is accompanied by a low-affinity → high-affinity change at the agonist-binding sites. The fact that the gating conformational change of muscle AChRs can be kinetically modeled as a two-state reaction has paved the way to the experimental characterization of the corresponding transition state, which represents a snapshot of the continuous sequence of molecular events separating the closed and open states. Previous studies of fully (di) liganded AChRs, combining single-channel kinetic measurements, site-directed mutagenesis, and data analysis in the framework of the linear free-energy relationships of physical organic chemistry, have suggested a transition-state structure that is consistent with channel opening being an asynchronous conformational change that starts at the extracellular agonistbinding sites and propagates toward the intracellular end of the pore. In this paper, I characterize the gating transition state of unliganded AChRs, and report a remarkable difference: unlike that of diliganded gating, the unliganded transition state is not a hybrid of the closed- and open-state structures but, rather, is almost indistinguishable from the open state itself. This displacement of the transition state along the reaction coordinate obscures the mechanism underlying the unliganded closed ⇋ open reaction but brings to light the malleable nature of free-energy landscapes of ion-channel gating. [ABSTRACT FROM AUTHOR]

The appearance of viral strains that are resistant to protease inhibitors is one of the most serious problems in the chemotherapy of HIV-1/ADDS. The most pervasive drug-resistant mutants are those that affect all inhibitors in clinical use. In this paper, we have characterized a multiple-drug-resistant mutant of the HIV-1 protease that affects indinavir, nelfinavir, saquinavir, ritonavir, amprenavir, and lopinavir. This mutant (MDR-HM) contains six amino acid mutations (L10I/M46I/I54V/V82A/I84V/ L90M) located within and outside the active site of the enzyme. Microcalorimetric and enzyme kinetic measurements indicate that this mutant lowers the affinity of all inhibitors by 2-3 orders of magnitude. By comparison, the multiiple-drug-resistant mutant only increased the K[subm] of the substrate by a factor of 2, indicating that the substrate is able to adapt to the changes caused by the mutations and maintain its binding affinity. To understand the origin of resistance, three submutants containing mutations in specific regions were also studied, i.e., the active site (V82A/I84V), flap region (M46I/I54V), and dimerization region (L10I/L90M). None of these sets of mutations by themselves lowered the affinity of inhibitors by more than 1 order of magnitude, and additionally, the sum of the effects of each set of mutations did not add up to the overall effect, indicating the presence of cooperative effects. A mutant containing only the four active site mutations (V82A/I84V/M46I/I54V) only showed a small cooperative effect, suggesting that the mutations at the dimer interface (L10I/L90M) play a major role in eliciting a cooperative response. These studies demonstrate that cooperative interactions contribute an average of 1.2 ± 0.7 kcal/mol to the overall resistance, most of the cooperative effect (0.8 ± 0.7 kcal/mol) being mediated by the mutations at the dimerization interface. Not all inhibitors in clinical use are affected the same by long-range cooperative interactions between mutations. These interactions can amplify the effects of individual mutations by factors ranging between 2 and 40 depending on the inhibitor. Dissection of the energetics of drug resistance into enthalpic and entropic components provides a quantitative account of the inhibitor response and a set of thermodynamic guidelines for the design of inhibitors with a lower susceptibility to this type of mutations. [ABSTRACT FROM AUTHOR]

The SecA ATPase motor is a central component of the eubacterial protein translocation machinery. It is comprised of N- and C-domain substructures, where the N-domain is comprised of two nucleotide-binding domains that flank a preprotein-binding domain (PPXD), while the C-domain binds phospholipids as well as SecB chaperone. Our recent crystal structure of Bacillus subtilis SecA protomer [Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026] along with experimental support for the correct dimer structure [Ding, H., Hunt, J. F., Mukerji, I., and Oliver, D. (2003) Biochemistry 42, 8729-8738] have now allowed us to study SecA structural dynamics during interaction with various translocation ligands and to relate these findings to current models of SecA-dependent protein translocation. In this paper, we utilized fluorescence resonance energy transfer methodology with genetically engineered SecA proteins containing unique pairs of tryptophan and fluorophore-labeled cysteine residues within the PPXD and C-domains of SecA to investigate the interaction of these two domains and their response to temperature, model membranes, and nucleotide. Consistent with the crystal structure of SecA, we found that the PPXD and C-domains are proximal to one another in the ground state. Increasing temperature or binding to model membranes promoted a loosening of PPXD and C-domain association, while ADP binding promoted a tighter association. A similar pattern of PPXD and C-domain association was obtained also for Escherichia coli SecA protein. Furthermore, a hyperactive Azi-PrlD SecA protein of E. coli had increased PPXD and C-domain separation, consistent with its activation in the ground state. Interestingly, PPXD and C-domain separation occurred prior to the onset of major temperature-induced conformational changes in both the PPXD and C-domains of SecA. Our results support a model in which PPXD and C-domain proximity is important for regulating the initial stages of SecA activation, and they serve also as a template for future structural studies aimed at elucidation of the chemomechanical cycle of SecA-dependent protein translocation. [ABSTRACT FROM AUTHOR]

Solution structure of the region 4 of σ[sup70] subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain [sup13]C resonances for a recombinant Rise-tagged protein containing the whole region 4 and a part of region 3.2 of σ[sup70] in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the [sup13]Cα, [sup13]CO, and [sup1]Hα nuclei and of vicinal [sup3]J[subHNHα] coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and [sup3]J[subHNHα] couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the σ[supA] subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and σ[sup70] of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R[sub1] and R[sub2], and {[sup1]H}-[sup15]N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of σ[sup70][sub4] protein in its protonated state. [ABSTRACT FROM AUTHOR]

δ-Atracotoxin-Arla (δ-ACTX-Arla) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion a-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing δ-ACTX-Arla. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHC1 and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. [SUP1]H NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic δ-ACTX-Arla produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that δ-ACTX-Arla produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic δ-ACTX-Arla paves the way for future structure-activity studies to determine the toxin pharmacophore. [ABSTRACT FROM AUTHOR]

In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the α-spectrin SH3 domain. This methodology includes the measurement of multiple [sup 13]C-[sup 13]C distance restraints on biosynthetically site-directed [sup 13]C-enriched samples, obtained by growing bacteria on [2-[sup 13]C]glycerol and [1,3-[sup 13]C]glycerol. 3D [sup 15]N-[sup 13]C--[sup 13]C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C[sup α]-C[sup α], CO-CO, C[sup α]-CO, and CO-C[sup α] type appear. Additional restraints for confining the structure were obtained from φ and ψ backbone torsion angles of 29 residues derived from C[sup α], C[sup β], CO, NH, and H[sup α] chemical shifts. Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 Å with respect to the average structure. [ABSTRACT FROM AUTHOR]

The roles of a pair of conserved positively charged residues R82 and R92 at a catalytic loop of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) have been investigated by site-directed mutagenesis and biochemical analysis. In the structure of HPPK in complex with ATP and a 6-hydroxymethyl-7,8-dihydropterin (HP) analogue, the guanidinium group of R82 forms two hydrogen bonds with the α-phosphate and that of R92 two hydrogen bonds with the β-phosphate, In the structure of HPPK in complex with α,β-methyleneadenosine triphosphate (AMPCPP, an ATP analogue) and HP, the guanidinium group of R82 has no direct interaction with AMPCPP and that of R92 forms two hydrogen bonds with the α-phosphate. Substitution of R82 with alanine caused a decrease in the rate constant for the chemical step by a factor of ∼380, but there were no significant changes in the binding energy or binding kinetics of either substrate. Substitution of R92 with alanine caused a decrease in the rate constant for the chemical step by a factor of ∼3.5 × 10[sup 4]. The mutation caused no significant changes in the binding energy or binding kinetics of MgATP. It did not cause a significant change in the binding energy of HP either but caused a decrease in the association rate constant for the binding of HP by a factor of ∼4.5 and a decrease in the dissociation rate constant by a factor of ∼10, The overall structures of the ternary complexes of both mutants were very similar to the corresponding structure of wild-type HPPK as described in the companion paper. The results suggest that R82 does not contribute to the binding of either substrate, and R92 is dispensable for the binding of MgATP but plays a role in facilitating the binding of HP. Both R82 and R92 are important for catalysis, and R92 plays a critical role in the transition state stabilization. [ABSTRACT FROM AUTHOR]

Presents a paper which describes the overexpression of L-ribulose-5-phosphate 4-epimerase at a very high level, its purification, and the preparation of site-directed mutants in which the putative metal ion ligands are modified. Observation of low levels of aldolase activity; Indepth look at the experimental procedures conducted in the study; Preparation and characterization of mutant enzymes; Results of the study.