Biomedical subjects

Yingchun Li

Publications and source records attributed to Yingchun Li.

15 recordsLinked to original sources

PubMed2006

p53 and p73 display common and distinct requirements for sequence specific binding to DNA.

Although p53 and p73 share considerable homology in their DNA-binding domains, there have been few studies examining their relative interactions with DNA as purified proteins. Comparing p53 and p73beta proteins, our data show that zinc chelation by EDTA is significantly more detrimental to the ability of p73beta than of p53 to bind DNA, most likely due to the greater effect that the loss of zinc has on the conformation of the DNA-binding domain of p73. Furthermore, prebinding to DNA strongly protects p73beta but not p53 from chelation by EDTA suggesting that DNA renders the core domain of p73 less accessible to its environment. Further exploring these biochemical differences, a five-base sub-sequence was identified in the p53 consensus binding site that confers a greater DNA-binding stability on p73beta than on full-length p53 in vitro. Surprisingly, p53 lacking its C-terminal non-specific DNA-binding domain (p53Delta30) demonstrates the same sequence discrimination as does p73beta. In vivo, both p53 and p73beta exhibit higher transactivation of a reporter with a binding site containing this sub-sequence, suggesting that lower in vitro dissociation translates to higher in vivo transactivation of sub-sequence-containing sites.

Base Sequence↗

PubMed2006

Predicting substrates by docking high-energy intermediates to enzyme structures.

With the emergence of sequences and even structures for proteins of unknown function, structure-based prediction of enzyme activity has become a pragmatic as well as an interesting question. Here we investigate a method to predict substrates for enzymes of known structure by docking high-energy intermediate forms of the potential substrates. A database of such high-energy transition-state analogues was created from the KEGG metabolites. To reduce the number of possible reactions to consider, we restricted ourselves to enzymes of the amidohydrolase superfamily. We docked each metabolite into seven different amidohydrolases in both the ground-state and the high-energy intermediate forms. Docking the high-energy intermediates improved the discrimination between decoys and substrates significantly over the corresponding standard ground-state database, both by enrichment of the true substrates and by geometric fidelity. To test this method prospectively, we attempted to predict the enantioselectivity of a set of chiral substrates for phosphotriesterase, for both wild-type and mutant forms of this enzyme. The stereoselectivity ratios of the six enzymes considered for those four substrate enantiomer pairs differed over a range of 10- to 10,000-fold and underwent 20 switches in stereoselectivities for favored enantiomers, compared to the wild type. The docking of the high-energy intermediates correctly predicted the stereoselectivities for 18 of the 20 substrate/enzyme combinations when compared to subsequent experimental synthesis and testing. The possible applications of this approach to other enzymes are considered.

Algorithms↗

PubMed2006

Resolution of chiral phosphate, phosphonate, and phosphinate esters by an enantioselective enzyme library.

An array of 16 enantiomeric pairs of chiral phosphate, phosphonate, and phosphinate esters was used to establish the breadth of the stereoselective discrimination inherent within the bacterial phosphotriesterase and 15 mutant enzymes. For each substrate, the leaving group was 4-hydroxyacetophenone while the other two groups attached to the phosphorus core consisted of an asymmetric mixture of methyl, methoxy, ethyl, ethoxy, isopropoxy, phenyl, phenoxy, cyclohexyl, and cyclohexoxy substituents. For the wild-type enzyme, the relative rates of hydrolysis for the two enantiomers ranged from 3 to 5.4 x 10(5). Various combinations of site-specific mutations within the active site were used to create modified enzymes with alterations in their enantioselective properties. For the single-site mutant enzyme, G60A, the stereoselectivity is enhanced relative to that of the wild-type enzyme by 1-3 orders of magnitude. Additional mutants were obtained where the stereoselectivity is inverted relative to the wild-type enzyme for 13 of the 16 pairs of enantiomers tested for this investigation. The most dramatic example was obtained for the hydrolysis of 4-acetylphenyl methyl phenyl phosphate. The G60A mutant preferentially hydrolyzes the SP-enantiomer by a factor of 3.7 x 10(5). The I106G/F132G/H257Y mutant preferentially hydrolyzes the RP-enantiomer by a factor of 9.7 x 10(2). This represents an enantioselective discrimination of 3.6 x 10(8) between these two mutants, with a total of only four amino acid changes. The rate differential between the two enantiomers for any given mutant enzyme is postulated to be governed by the degree of nonproductive binding within the enzyme active site and stabilization of the transition state. This hypothesis is supported by computational docking of the high-energy, pentavalent form of the substrates to modeled structures of the mutant enzyme; the energies of the docked transition-state analogues qualitatively capture the enantiomeric preferences of the various mutants for the different substrates. These results demonstrate that the catalytic properties of the wild-type phosphotriesterase can be exploited for the kinetic resolution of a wide range of phosphate, phosphonate, and phosphinate esters and that the active site of this enzyme is remarkably amenable to structural perturbations via amino acid substitution.

Amino Acid Substitution↗

PubMed2006

Uronate isomerase: a nonhydrolytic member of the amidohydrolase superfamily with an ambivalent requirement for a divalent metal ion.

Uronate isomerase, a member of the amidohydrolase superfamily, catalyzes the isomerization of D-glucuronate and D-fructuronate. During the interconversion of substrate and product the hydrogen at C2 of D-glucuronate is transferred to the pro-R position at C1 of the product, D-fructuronate. The exchange of the transferred hydrogen with solvent deuterium occurs at a rate that is 4 orders of magnitude slower than the interconversion of substrate and product. The enzyme catalyzes the elimination of fluoride from 3-deoxy-3-fluoro-D-glucuronate. These results have been interpreted to suggest a chemical reaction mechanism in which an active site base abstracts the proton from C2 of D-glucuronate to form a cis-enediol intermediate. The conjugate acid then transfers this proton to C1 of the cis-enediol intermediate to form D-fructuronate. The loss of fluoride from 3-deoxy-3-fluoro-D-glucuronate is consistent with a stabilized carbanion at C2 of the substrate during substrate turnover. The slow exchange of the transferred hydrogen with solvent water is consistent with a shielded conjugate acid after abstraction of the proton from either D-glucuronate or D-fructuronate during the isomerization reaction. This conclusion is supported by the competitive inhibition of the enzymatic reaction by D-arabinaric acid and the monohydroxamate derivative with Ki values of 13 and 670 nM, respectively. There is no evidence to support a hydride transfer mechanism for uronate isomerase. The wild type enzyme was found to contain 1 equiv of zinc per subunit. The divalent cation could be removed by dialysis against the metal chelator, dipicolinate. However, the apoenzyme has the same catalytic activity as the Zn-substituted enzyme and thus the divalent metal ion is not required for enzymatic activity. This is the only documented example of a member in the amidohydrolase superfamily that does not require one or two divalent cations for enzymatic activity.

Aldose-Ketose Isomerases↗

PubMed2006

Preparation and evaluation of uniform-size (-)-ephedrine-imprinted polymeric microspheres by multi-step swelling and suspension polymerization.

Ephedrine-imprinted polymeric microspheres have been prepared in an aqueous system by multi-step swelling and suspension polymerization, using methacrylic acid (MAA) as a functional monomer, and ethylene glycol dimethacrylate (EGDMA) as a cross-linker. Scanning electron microscopy (SEM) was used as a means to identify the structure features of the obtained polymers. Further, we examined the recognition mechanism of the polymers and the influences of some chromatographic conditions, such as the mobile-phase composition, flow-rate, column temperature and sample amount on the retentivity and selectivity for (-)-ephedrine and (+)-ephedrine. The results reveal that stable macroporous polymer beads with good size monodispersity were obtained, the average size of which was 3-5 microm. Baseline chiral separation of the template isomers was achieved on a short column (50 mmx4.6 mm i.d.) when the prepared polymer beads were used as a stationary phase, while the non-imprinted polymers (NIPs) did not show such ability. The optimized chromatographic condition was as follows: acetonitrile-acetic acid (99.8/0.2, v/v) as the mobile phase; sample amount, 40-80 microg; flow rate, 1.0 ml min-1; and column temperature, room temperature, respectively. It is assumed that two classes of binding sites exist in the porous polymers, one being hydrophilic binding sites, the other being hydrophobic binding sites.

Acetic Acid↗

PubMed2005

Inhibitors designed for the active site of dihydroorotase.

Four new compounds have been synthesized as potential inhibitors of dihydroorotase from Escherichia coli. NMR spectroscopy was used to show that 4,6-dioxo-piperidine-2(S)-carboxylic acid (3), exists in solution as a mixture of the hydrate (7), enol (8), and enolate (9) tautomeric forms. This compound was found to be a competitive inhibitor versus dihydroorotate and thio-dihydroorotate at pH values of 7-9. The K(i) of 76 microM was lowest at pH7.0 where the ketone and hydrate forms of the inhibitor 3 predominate in solution. Compound 3 was reduced to the two diastereomeric 4-hydroxy derivatives (4 and 5) and then dehydrated to yield the alkene derivative, 1,2,3,6-tetrahydro-6-oxopyridine-2(S)-carboxylic acid (6). Compounds 4-6 were competitive inhibitors versus thio-dihydroorotate at pH 8.0 with K(i) values of 3.0, 1.6, and 2.3 mM. Dihydroorotase was unable to dehydrate the 4-hydroxy derivative 4 or 5 to the alkene 6 or catalyze the reverse reaction.

Binding Sites↗

PubMed2005

Inner core differential motion confirmed by earthquake waveform doublets.

We analyzed 18 high-quality waveform doublets with time separations of up to 35 years in the South Sandwich Islands region, for which the seismic signals have traversed the inner core as PKP(DF). The doublets show a consistent temporal change of travel times at up to 58 stations in and near Alaska, and they show a dissimilarity of PKP(DF) coda. Using waveform doublets avoids artifacts of earthquake mislocations and contamination from small-scale heterogeneities. Our results confirm that Earth's inner core is rotating faster than the mantle and crust at about 0.3 degrees to 0.5 degrees per year.

Journal Article↗

PubMed2004

Mechanism of the dihydroorotase reaction.

Dihydroorotase (DHO) is a zinc metalloenzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. A chemical mechanism was proposed on the basis of an analysis of the effects of pH, metal substitution, solvent isotope effects, mutant proteins, and alternative substrates on the enzyme-catalyzed reaction. The pH-rate profiles for the hydrolysis of dihydroorotate or thiodihydroorotate demonstrated that a single group from the enzyme must be unprotonated for maximal catalytic activity. Conversely, the pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or cadmium by reconstitution of the apoenzyme with divalent cations in the presence of bicarbonate. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of the residue (Asp-250) that hydrogen bonds to the bridging hydroxide (or water) resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp-250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during hydrolysis of dihydroorotate. The X-ray crystal structure of DHO showed that the exocyclic alpha-carboxylate of dihydroorotate is bound to the protein via electrostatic interactions with Arg-20, Asn-44, and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thio analogue of dihydroorotate was found to be a good substrate of the enzyme. A comprehensive chemical mechanism for DHO was proposed on the basis of the experimental findings in this study and the X-ray crystal structure.

Deuterium↗

PubMed2004

Catalytic properties of the PepQ prolidase from Escherichia coli.

The PepQ prolidase from Escherichia coli catalyzes the hydrolysis of dipeptide substrates with a proline residue at the C-terminus. The pepQ gene has been cloned, overexpressed, and the enzyme purified to homogeneity. The k(cat) and k(cat)/K(m) values for the hydrolysis of Met-Pro are 109 s(-1) and 8.4 x 10(5)M(-1)s(-1), respectively. The enzyme also catalyzes the stereoselective hydrolysis of organophosphate triesters and organophosphonate diesters. A series of 16 organophosphate triesters with a p-nitrophenyl leaving group were assessed as substrates for PepQ. The S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was hydrolyzed with a k(cat) of 36 min(-1) and a k(cat)/K(m) of 710 M(-1)s(-1). The corresponding R(P)-enantiomer was hydrolyzed more slowly with a k(cat) of 0.4 min(-1) and a k(cat)/K(m) of 11 M(-1)s(-1). The PepQ prolidase can be utilized for the kinetic resolution of racemic phosphate esters. The PepQ prolidase was shown to hydrolyze the p-nitrophenyl analogs of the nerve agents GB (sarin), GD (soman), GF, and VX.

Base Sequence↗

PubMed2004

Enzymatic resolution of chiral phosphinate esters.

The bacterial phosphotriesterase has been shown to catalyze the stereoselective hydrolysis of phosphinate esters. The wild-type enzyme preferentially hydrolyzes the SP-enantiomers of methyl phenyl p-X-phenylphosphinate esters by 3 orders of magnitude. The mutant enzyme, I106T/F132A/H254G/H257W, exhibits the opposite stereoselectivity and hydrolyzes the RP-enantiomer up to 30 times faster than the corresponding SP-enantiomer. The enantiomerically pure phosphinate esters, prepared from the kinetic resolution of racemic mixtures, can serve as the entry point for the chemoenzymatic preparation of P-chiral phosphines and phosphine oxides.

Esters↗

PubMed2004

Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase.

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.

Aryldialkylphosphatase↗

PubMed2003

Operational control of stereoselectivity during the enzymatic hydrolysis of racemic organophosphorus compounds.

The wild-type bacterial phosphotriesterase catalyzes the stereoselective hydrolysis of racemic pairs of organophosphorus compounds. The enzymatic stereoselectivity can be substantially enhanced via systematic alteration of the pKa for the leaving group phenol in the target substrates. These changes alter the rate-limiting step for substrate turnover from a diffusional event to phosphorus-oxygen bond cleavage. Turnover ratios in excess of 5000:1 were achieved using phenols with pKa values greater than 8.5. This method has enabled the isolation of the RP-enantiomer of 4-acetylphenyl methyl phenylphosphonate with an enantiomeric excess of >99% via a kinetic resolution of the racemate.

Aryldialkylphosphatase↗

PubMed2003

Organophosphate nerve agent toxicity in Hydra attenuata.

The toxicity for analogues of sarin (GB), soman (GD), and VX was evaluated using Hydra attenuata as a model organism. The organophosphate nerve agent analogue simulants used in this investigation included the following: isopropyl p-nitrophenyl methylphosphonate (for GB); pinacolyl p-nitrophenyl methylphosphonate (for GD); and diisopropyl S-(2-diisopropylaminoethyl)phosphorothioate, diethyl S-(2-diisopropylaminoethyl)phosphorothioate, and diethyl S-(2-trimethylaminoethyl)phosphorothioate (for VX). The toxicity of each organophosphate nerve agent was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There is a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate chemical warfare agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogues was 2 orders of magnitude more toxic than the analogue for GD and 4 orders of magnitude more toxic than the analogue for GB.

Administration, Oral↗

PubMed2003

Schizosaccharomyces pombe Ras1 effector, Scd1, interacts with Klp5 and Klp6 kinesins to mediate cytokinesis.

Fission yeast Scd1 is an exchange factor for Cdc42 and an effector of Ras1. In a screen for scd1 interacting genes, we isolated klp5 and klp6, which encode presumptive kinesins. Klp5 and Klp6 form a complex to control the same processes, which so far include microtubule dynamics and chromosome segregation. We showed that klp5 or klp6 inactivation in combination with the scd1 deletion (scd1delta) created a synthetic temperature-dependent growth defect. Further genetic analysis demonstrated that Klp5 and Klp6 interacted specifically with the Ras1-Scd1 pathway, but not with the Ras1-Byr2 pathway. In addition, Klp5 and Klp6 can stably associate with Scd1 and Cdc42. A deletion in the Scd1 C terminus, which contains the PB1 domain, prevented Scd1 binding to Klp5/6 and caused a growth defect in Klp5/6 mutant cells that is indistinguishable from that induced by scd1delta. Analysis of the double-mutant phenotype indicated that at the nonpermissive temperature, cells failed to undergo cytokinesis efficiently. These cells contained abnormal contractile rings in which F-actin and Mid1, a key regulator of F-actin ring formation and positioning, are mispositioned and fragmented. These data suggest that Klp5/6 cooperate with the Ras1-Scd1 pathway to influence proper formation of the contractile ring for cytokinesis.

Actins↗

PubMed2002

Enzymatic synthesis of chiral organophosphothioates from prochiral precursors.

The phosphotriesterase from Pseudomonas diminuta has been shown to selectively cleave the pro-R p-nitrophenolate substituent from bis-p-nitrophenyl alkyl phosphothioate esters. When the alkyl substituent is methyl, ethyl, or isopropyl the enantiomeric excess of the product is >/=99%. Manipulation of the active site through mutagenesis has enabled the preparation of protein variants that preferentially hydrolyze the pro-S substituent of the target substrates. This methodology thus permits the preparation of chiral products from prochiral precursors.

Aryldialkylphosphatase↗