The ultrafast reactions in the photochromic cycle of water-soluble fulgimide photoswitches.

Photochromic switches are essential for the control and manipulation of nanoscale reactions and processes. The expansion of their application to aqueous environments depends strongly on the development of optimized water-soluble photoswitches. Here we present a femtosecond time-resolved investigation of the photochromic reactions (transition between the open and the closed form) of a water-soluble indolylfulgimide. We observe a pronounced effect of the protic nature of water as a solvent on the ultrafast ring-opening reaction. Typically, the excited state of the closed form has a larger dipole moment than the ground state, which leads to stabilization of the excited state in polar solvents and hence a lifetime (3 ps) longer than in non-polar solvents (2 ps). However, in water, despite the increased solvent polarity and the increased excited state dipole moment, the opposite trend for the excited state lifetime is observed (1.8 ps). This effect is caused by the opening of a new excited state deactivation pathway involving proton transfer reactions.

The versatility of phosphoenolpyruvate and its vinyl ether products in biosynthesis.

The diverse enzymes that use phosphoenolpyruvate as a substrate lie at the heart of cellular energy metabolism, as well as a number of critical biosynthetic pathways. The versatility of the enol ether linkage is reflected not only in the rich chemistry and enzymology of PEP, but also in the variety of metabolites in which the high-energy enol ether linkage is preserved.

Polyacrylamides bearing pendant alpha-sialoside groups strongly inhibit agglutination of erythrocytes by influenza A virus: multivalency and steric stabilization of particulate biological systems.

An alpha-sialoside linked to acrylamide by a short connector (5-acetamido-2-O-(N-acryloyl-8-amino-5-oxaoctyl)-2,6-anhydro-3,5-d ideoxy-D-galacto-alpha-nonulopyranosonoic acid, 1) was prepared. Compound 1 formed high molecular weight copolymers with acrylamide, derivatives of acrylamide, and/or vinylpyrrolidone upon photochemically-initiated free radical polymerization. Those copolymers for which the substituents on the acrylamido nitrogen were small inhibited the agglutination of chicken erythrocytes induced by influenza virus (X-31 (H3N2); a recombinant strain of A/Aichi/2/68 (H3N2) and A/Puerto Rico/8/34 grown in chicken eggs). The inhibitory power of the polymers depended strongly on the conditions of polymerization and the sialic acid content of the polymer. The strongest inhibitors were copolymers (poly(1-co-acrylamide)) formed from mixtures of monomer containing [1]/([1] + [acrylamide]) approximately 0.2-0.7; these copolymers inhibited hemagglutination 10(4)-10(5) times more strongly than did similar concentrations of alpha-methyl sialoside (calculated on the basis of the total concentration of individual sialic acid groups in the solution, whether attached to polymer or present as monomers). Samples polymerized in the presence of low concentrations of cross-linking reagents (bis(acrylamido)methane, BIS, and 2,2'-bis(acrylamido)ethyl disulfide, BAC) also showed increased inhibition (10-10(3)-fold relative to monomers), but their use was limited by their poor solubility. Sterically demanding substituents on any position of the acrylamide component (substituents attached to the vinyl group or N-alkyl groups that are larger than hydroxyethyl) reduced the inhibitory power of the polymer. A 1H NMR assay and a fluorescence depolarization assay showed that poly(1-co-acrylamide) bound to a solubilized trimeric form of the viral receptor for sialic acid (bromelain cleaved hemagglutinin, BHA), less tightly than 1, on a per sialic acid basis. A similar result was also obtained with a model system comprising lactic dehydrogenase (a tetramer) and polymeric derivatives of oxamic acid: that is, poly((28, 29, 30, or 31)-co-acrylamide) had a higher inhibition constant for tetrameric lactic dehydrogenase than did the corresponding monomers (28, 29, 30, or 31) on a per oxamate basis. Poly(1-co-acrylamide) is, in principle, capable of inhibiting the agglutination of erythrocytes by several mechanisms: (1) entropically enhanced binding of the polymer (acting as a polyvalent inhibitor) to the surface of the virus; (2) steric interference of the approach of the virus to the surface of the erythrocyte by a water-swollen layer of the polymer on the surface of the virus; (3) aggregation of the virus induced by the polymer.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of aromatic thiols on thiol-disulfide interchange reactions that occur during protein folding.

The folding of disulfide containing proteins from denatured protein to native protein involves numerous thiol-disulfide interchange reactions. Many of these reactions include a redox buffer, which is a mixture of a thiol (RSH) and the corresponding disulfide (RSSR). The relationship between the structure of RSH and its efficacy in folding proteins in vitro has been investigated only to a limited extent. Reported herein are the effects of aliphatic and especially aromatic thiols on reactions that occur during protein folding. Aromatic thiols may be particularly efficacious as their thiol pK(a) values and reactivities match those of the in vivo catalyst, protein disulfide isomerase (PDI). This investigation correlates the thiol pK(a) values of aromatic thiols with their reactivities toward small molecule disulfides and the protein insulin. The thiol pK(a) values of nine para-substituted aromatic thiols were measured; a Hammett plot constructed using sigma(p-) values yielded rho = -1.6 +/- 0.1. The reactivities of aromatic and aliphatic thiols with 2-pyridyldithioethanol (2-PDE), a small molecule disulfide, were determined. A plot of reactivity versus pK(a) of the aromatic thiols had a slope (beta) of 0.9. The ability of these thiols to reduce (unfold) the protein insulin correlates strongly with their ability to reduce 2-PDE. Since the reduction of protein disulfides occurs during protein folding to remove mismatched disulfides, aromatic thiols with high pK(a) values are expected to increase the rate not only of protein unfolding but protein folding as well.

Using affinity capillary electrophoresis to determine binding stoichiometries of protein-ligand interactions.

We have developed a new method utilizing affinity capillary electrophoresis (ACE) for the determination of binding stoichiometries in biochemical systems. Using the same concentration of a ligand in the sample and the electrophoresis buffer, the appearance of an inverted peak corresponding to the free ligand in the resulting electropherogram provides a criterion of binding of a ligand to its receptor protein. For both low (fast off rates) and high (slow off rates) affinity systems, analysis of the integration of free ligand peak in electropherograms as a function of the total concentration of a ligand in samples at constant concentration of receptor protein yields the binding stoichiometry of the ligand to the protein. Applications of this technique to studies of (i) the inhibition of carbonic anhydrases (CA, EC 4.2.1.1, from human and bovine erythrocytes) by 4-alkylbenzenesulfonamide 1, (ii) the interaction of a monoclonal antibody to human serum albumin (anti-HSA) with its antigen HSA, and (iii) the binding of streptavidin (from Streptomyces avidinii) to biotin derivatives (monobiotinylated oligodeoxyribonucleotide 2, fluorescein biotin, or Lucifer Yellow biotin) yield stoichiometries of 1:1, 1:2, and 1:4, respectively. For multivalent, tight-binding systems, this ACE method can readily separate stable intermediate species. This method is generally applicable to both tight- and weak-binding systems, requires only nanograms of proteins and ligands, involves no radioactive materials, and does not require changes in electrophoretic mobilities of receptor proteins upon binding with ligands. It thereby provides a rapid, sensitive, and convenient method for measuring binding stoichiometries of ligands to proteins.

Stereochemical course of enzymatic enolpyruvyl transfer and catalytic conformation of the active site revealed by the crystal structure of the fluorinated analogue of the reaction tetrahedral intermediate bound to the active site of the C115A mutant of MurA.

MurA (UDP-GlcNAc enolpyruvyl transferase), the first enzyme in bacterial peptidoglycan biosynthesis, catalyzes the enolpyruvyl transfer from phosphoenolpyruvate (PEP) to the 3'-OH of UDP-GlcNAc by an addition-elimination mechanism that proceeds through a tetrahedral ketal intermediate. The crystal structure of the Cys115-to-Ala (C115A) mutant of Escherichia coli MurA complexed with a fluoro analogue of the tetrahedral intermediate revealed the absolute configuration of the adduct and the stereochemical course of the reaction. The fluorinated adduct was generated in a preincubation of wild-type MurA with (Z)-3-fluorophosphoenolpyruvate (FPEP) and UDP-GlcNAc and purified after enzyme denaturation. The fluorine substituent stabilizes the tetrahedral intermediate toward decomposition by a factor of 10(4)-10(6), facilitating manipulation of the adduct. The C115A mutant of MurA was utilized to avoid the microheterogeneity that arises in the wild-type MurA from the attack of Cys115 on C-2 of FPEP in competition with the formation of the fluorinated adduct. The crystal structure of the complex was determined to 2.8 A resolution, and the absolute configuration at C-2 of the adduct was found to be 2R. Thus, addition of the 3'-OH of UDP-GlcNAc is to the 2-si face of FPEP, corresponding to the 2-re face of PEP. Given the previous observation that, in D2O, the addition of D+ to C-3 of PEP proceeds from the 2-si face [Kim, D. H., Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381], the addition across the double bond of PEP is anti. Also, because the overall stereochemical course has been shown to be either anti/syn or syn/anti [Lees, W. J., and Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 7329-7337], it now follows that the stereochemistry of elimination of H+ from C-3 and Pi from C-2 of the tetrahedral intermediate of the reaction is syn.

Ionic blockade of the rat connexin40 gap junction channel by large tetraalkylammonium ions.

The rat connexin40 gap junction channel is permeable to monovalent cations including tetramethylammonium and tetraethylammonium ions. Larger tetraalkyammonium (TAA(+)) ions beginning with tetrabutylammonium (TBA(+)) reduced KCl junctional currents disproportionately. Ionic blockade by tetrapentylammonium (TPeA(+)) and tetrahexylammonium (THxA(+)) ions were concentration- and voltage-dependent and occurred only when TAA(+) ions were on the same side as net K(+) efflux across the junction, indicative of block of the ionic permeation pathway. The voltage-dependent dissociation constants (K(m)(V(j))) were lower for THxA(+) than TPeA(+), consistent with steric effects within the pore. The K(m)-V(j) relationships for TPeA(+) and THxA(+) were fit with different reaction rate models for a symmetrical (homotypic) connexin gap junction channel and were described by either a one- or two-site model that assumed each ion traversed the entire V(j) field. Bilateral addition of TPeA(+) ions confirmed a common site of interaction within the pore that possessed identical K(m)(V(j)) values for cis-trans concentrations of TPeA(+) ions as indicated by the modeled I-V relations and rapid channel block that precluded unitary current measurements. The TAA(+) block of K(+) currents and bilateral TPeA(+) interactions did not alter V(j)-gating of Cx40 gap junctions. N-octyl-tributylammonium and -triethylammonium also blocked rCx40 channels with higher affinity and faster kinetics than TBA(+) or TPeA(+), indicative of a hydrophobic site within the pore near the site of block.

(E)-enolbutyryl-UDP-N-acetylglucosamine as a mechanistic probe of UDP-N-acetylenolpyruvylglucosamine reductase (MurB).

UDP-N-acetylenolpyruvylglucosamine reductase (MurB), a peptidoglycan biosynthetic enzyme from Escherichia coli, reduces both (E)- and (Z)-isomers of enolbutyryl-UDP-GlcNAc, C4 analogs of the physiological C3 enolpyruvyl substrate, to UDP-methyl-N-acetylmuramic acid in the presence of NADPH. The X-ray crystal structure of the (E)-enolbutyryl-UDP-GlcNAc-MurB complex is similar to that of the enolpyruvyl-UDP-GlcNAc-MurB complex. In both structures the groups thought to be involved in hydride transfer to C3 and protonation at C2 of the enol ether substrate are arranged anti relative to the enol double bond. The stereochemical outcome of reduction of (E)-enolbutyryl-UDP-GlcNAc by NADPD in D2O is thus predicted to yield a (2R,3R)-dideuterio product. This was validated by conversion of the 2,3-dideuterio-UDP-methyl-N-acetylmuramic acid product to 2,3-dideuterio-2-hydroxybutyrate, which was shown to be (2R) by enzymatic analysis and (3R) by NMR comparison to authentic (2R,3R)- and (2R,3S)-2,3-dideuterio-2-hydroxybutyrate. Remarkably, the (E)-enolbutyryl-UDP-GlcNAc was found to partition between reduction to UDP-methyl-N-acetylmuramic and isomerization to the (Z)-substrate isomer in the MurB active site, indicative of a C2 carbanion/enol species that is sufficiently long-lived to rotate around the C2-C3 single bond during catalysis.

Analysis of fluoromethyl group chirality establishes a common stereochemical course for the enolpyruvyl transfers catalyzed by EPSP synthase and UDP-GlcNAc enolpyruvyl transferase.

The stereochemistry of transient methyl group formation at C-3 of phosphoenolpyruvate (PEP) in the reaction catalyzed by 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase has been examined using the pseudosubstrates, (E)- and (Z)-3-fluorophosphoenolpyruvate (FPEP). Kinetically stable, chiral [1H, 2H]fluoromethyl analogs of the reaction tetrahedral intermediate were isolated and subjected to decomposition and stereochemical analysis. EPSP synthase was found to catalyze the 2-re face addition of solvent-derived hydrogen to C-3 of FPEP (corresponding to the 2-si face of PEP). Comparison of these data with prior analogous work on the MurA reaction [Kim, D.H., Lees, W.J., & Walsh, C. T. (1995) J. Am. Chem. Soc. 117, 6380-6381] suggests that the two enolpyruvyl transferases share a common stereochemical course, further strengthening the mechanistic, structural, and evolutionary relationship between the two enzymes.

Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli.

The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-acetylglucosamine [Mengin-Lecreulx, D., & van Heijenoort, J. (1994) J. Bacteriol. 176, 5788-5795], a fundamental precursor in bacterial peptidoglycan biosynthesis and the source of activated N-acetylglucosamine in lipopolysaccharide biosynthesis in Gram-negative bacteria. In the work described here, the GlmU protein and truncation variants of GlmU (N- and C-terminal) were purified and kinetically characterized for substrate specificity and reaction order. It was determined that the GlmU protein first catalyzed acetyltransfer followed by uridylyltransfer. The N-terminal portion of the enzyme was capable of only uridylyltransfer, and the C-terminus catalyzed only acetyltransfer. GlmU demonstrated a 12-fold kinetic preference (kcat/Km, 3.1 x 10(5) versus 2.5 x 10(4) L.mol-1.s-1) for acetyltransfer from acetyl-CoA to glucosamine-1-P as compared to UDP-glucosamine. No detectable uridylyltransfer from UTP to glucosamine-1-P was observed in the presence of GlmU; however, the enzyme was competent in catalyzing the formation of UDP-N-acetylglucosamine from UTP and N-acetylglucosamine-1-P (kcat/Km 1.2 x 10(6) L.mol-1.s-1). A two active site model for the GlmU protein was indicated both by domain dissection experiments and by assay of the bifunctional reaction. Kinetic studies demonstrated that a pre-steady-state lag in the production of UDP-N-acetylglucosamine from acetyl-CoA, UTP, and glucosamine-1-P was due to the release and accumulation of steady-state levels of the intermediate N-acetylglucosamine-1-P.

Characterization of a Cys115 to Asp substitution in the Escherichia coli cell wall biosynthetic enzyme UDP-GlcNAc enolpyruvyl transferase (MurA) that confers resistance to inactivation by the antibiotic fosfomycin.

The antibiotic fosfomycin inhibits bacterial cell wall biosynthesis by inactivation of UDP-GlcNAc enolpyruvyl tranferase (MurA). Prior work has established that Cys115 of Escherichia coli and Enterobacter cloacae MurA is the active site nucleophile alkylated by fosfomycin and implicated this residue in the formation of a covalent phospholactyl-enzyme adduct derived from substrate, phosphoenolpyruvate (PEP). On the basis of sequencing information from putative MurA homolog from Mycobacterium tuberculosis, we generated a C115D mutant of E. coli MurA that was highly active but fully resistant to time-dependent inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition by fosfomycin. Fosfomycin still bound to the active site of C115D MurA, as established by the observed reversible competitive inhibition vs PEP. In contrast to the broad pH-independent behavior of wild-type (WT) MurA, C115D mutant activity titrated across the pH range examined (pH 5.5-9) with an apparent pKa approximately 6, with kcatC115D ranging from approximately 10kcatWT at pH 5.5 to <0.1kcatWT at pH9.0. Km(PEP)115D was relatively constant in the pH range examined and increased approximately 100-fold relative to Km(PEP)WT. A fosfomycin-resistant C115E mutant with -1% activity of the C115D mutant was found to follow a pH dependence similar to that observed for C115D MurA. The contrasting pH dependences of WT and C115D MurA was also observed in the reaction with the pseudosubstrate, (Z)-3-fluorophosphoenolpyruvate, strongly suggesting a role for Cys/Asp115 as the general acid in the protonation of C-3 of PEP during MurA-catalyzed enol ether transfer. The difference in nucleophilicity between the carboxylate side chains of Asp115 and Glu115 and the thiolate group of Cys115 suggests that covalent enzyme adduct formation is not required for catalytic turnover and, furthermore, provides a chemical rationale for the resistance of the C115D and C115E mutants to fosfomycin inactivation.