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81.
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. 相似文献
82.
Summary. Two goals of multiple-dose factorial trials are (i) demonstrating improved effectiveness of a fixed combination over each of its components as well as (ii) identifying a safe and effective dose range. The authors address both goals though with focus on the second by closure procedures that guarantee strong control of the familywise error rate. Two different families of null hypotheses are investigated for bi-factorial dose–response designs that are monotone with respect to the matrix partial order. One is suitable to find the minimum effective dose(s) and the other one is large enough to identify the highest effective dose step(s). Likelihood ratio tests and appropriate multiple contrast tests are applied to an unbalanced clinical trial example taken from Hung (2000, Statistics in Medicine 19, 2079–2087). Full computer code written in the R language is available from the Internet. 相似文献
83.
The highly sensitive, convenient fluorescence assay, based on the oxidation of nonfluorescent 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) to highly fluorescent resorufin, is becoming increasingly popular for hydrogen peroxide quantitation. Yet, the intricacies of the horseradish peroxidase-catalyzed oxidation of the reductant substrate Amplex Red by hydrogen peroxide and the resulting resorufin could complicate the assay design and data interpretation. In particular, substrate inhibition and enzyme inactivation at higher hydrogen peroxide concentrations were known to affect the enzyme kinetics and end-point fluorescence. In addition, here we report the spontaneous transformation of resorufin to less or nonfluorescent product(s) in the absence of hydrogen peroxide and horseradish peroxidase. This spontaneous decay of resorufin fluorescence is most prominent in the pH range 6.2-7.7, likely due to general base-catalyzed de-N-acetylation and polymerization of resorufin. From a practical point of view, precautions for properly designing assays for hydrogen peroxide or characterizing hydrogen peroxide-generating systems are discussed based on the spontaneous transformation of resorufin to less fluorescent compound(s), substrate inhibition and enzyme inactivation at higher (>100 microM) hydrogen peroxide concentrations, and enzymatic oxidation of resorufin to nonfluorescent resazurin. 相似文献
84.
A fluorescent binding assay was developed to investigate the effects of mutagenesis on the binding affinity and substrate specificity of the chitin-binding domain of chitinase A1 from Bacillus circulans WL-12. The chitin-binding domain was genetically fused to the N-terminus of a green fluorescent protein, and the polyhistidine-tagged hybrid protein was expressed in Escherichia coli. Residues likely to be involved in the binding site were mutated and their contributions to binding and substrate specificity were evaluated by affinity electrophoresis and depletion assays. The experimental binding isotherms were analyzed by non-linear regression using a modified Langmuir equation. Non-conservative substitution of tryptophan residue (W687) nearly abolished chitin-binding affinity and dramatically lowered chitosan binding while retaining the original level of curdlan binding. Double mutation E668K/P689A had altered specificity for several substrates and also impaired chitin binding significantly. Other substitutions in the binding site altered substrate specificity but had little effect on overall affinity for chitin. Interestingly, mutation T682A led to a higher specificity towards chitinous substrates than the wildtype. Furthermore, the ChBD-GFP hybrid protein was tested for use in diagnostic staining of cell walls of fungi and yeast and for the detection of fungal infections in tissue samples. 相似文献
85.
Human hypoxanthine guanine phosphoribosyltransferase (HGPRT) lacks the ability to phosphoribosylate xanthine, a property exhibited by HGPRTs from many parasitic protozoa. Using random mutagenesis we have obtained a mutant, F36L, of human HGPRT that phosphoribosylates xanthine. Examination of the structure indicates that F36 does not make direct contact with the purine, but long-range modulation via loop IV, a segment contacting purine at C2 position, could influence substrate specificity. Expanded substrate specificity to include xanthine probably arises from increased flexibility of loop IV as a consequence of mutation at F36. Mutation of the corresponding residue, L44 in Plasmodium falciparum HGPRT, also results in alteration of K(m) and k(cat) for xanthine, substantiating its role in affecting purine base affinity. Our studies show that mutation of this residue in the core of the protein also affects the stability of both enzymes. 相似文献
86.
Janata J Holá K Kubala M Gakh O Parkhomenko N Matusková A Kutejová E Amler E 《Biochemical and biophysical research communications》2004,317(1):211-217
To determine whether the two domains of hepatitis C virus (HCV) NS3 and the NS4A interact with each other to regulate the RNA unwinding activity, this study compares the RNA unwinding, ATPase and RNA binding activities of three forms of NS3 proteins--the NS3H protein, containing only the helicase domain, the full-length NS3 protein, and the NS3-NS4A complex. The results revealed that NS3 displayed the weakest RNA helicase activity, not because it had lower ATPase or RNA binding activity than did NS3H or NS3-NS4A, but because it had the lowest RNA unwinding processivity. A mutant protein, R1487Q, which contained a mutation in the helicase domain, displayed a reduced protease activity as compared to the wild-type NS3-NS4A. Together, these results suggest the existence of interactions between the two domains of NS3 and the NS4A, which regulates the HCV NS3 protease and RNA helicase activities. 相似文献
87.
Georgieva D Rypniewski W Echner H Perbandt M Koker M Clos J Redecke L Bredehorst R Voelter W Genov N Betzel C 《Biochemical and biophysical research communications》2004,325(4):1406-1411
Proteinase K is widely used in tests for the presence of infectious prion protein causing fatal spongiform encephalopathies. To investigate possible interactions between the enzyme and the functionally important N-terminal prion domain, we crystallized mercury-inhibited proteinase K in the presence of the synthetic peptides GGGWGQPH and HGGGW. The octapeptide sequence is identical to that of a single octapeptide repeat (OPR) from the physiologically important OPR region. Here, we present the first direct evidence for the complex formation between a proteolytic enzyme and a segment of human prion molecule. The X-ray structures of the complexes at 1.4 and 1.8A resolution, respectively, revealed that in both cases the segment GGG is strongly bound as a real substrate at the substrate recognition site of the proteinase forming an antiparallel beta-strand between the two parallel strands of Asn99-Tyr104 and Ser132-Gly136. The complex is stabilized through an extended H-bonding network. 相似文献
88.
Höng JC Ivanov NV Hodor P Xia M Wei N Blevins R Gerhold D Borodovsky M Liu Y 《Journal of molecular biology》2004,337(2):307-317
We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains. 相似文献
89.
90.
Yoo A Seo YS Jung JW Sung SK Kim WT Lee W Yang DR 《Journal of structural biology》2006,156(3):407-420
The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe(2+)/H(2)O derived from Petunia hybrida has recently been established by X-ray crystallography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30-15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclusively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the positive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity. 相似文献