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Reduced Enzyme Activity (reduced + enzyme_activity)
Selected AbstractsSubstrate and inhibitor specificity of Mycobacterium avium dihydrofolate reductaseFEBS JOURNAL, Issue 13 2007Ronnie A. Böck Dihydrofolate reductase (EC 1.5.1.3) is a key enzyme in the folate biosynthetic pathway. Information regarding key residues in the dihydrofolate-binding site of Mycobacterium avium dihydrofolate reductase is lacking. On the basis of previous information, Asp31 and Leu32 were selected as residues that are potentially important in interactions with dihydrofolate and antifolates (e.g. trimethoprim), respectively. Asp31 and Leu32 were modified by site-directed mutagenesis, giving the mutants D31A, D31E, D31Q, D31N and D31L, and L32A, L32F and L32D. Mutated proteins were expressed in Escherichia coli BL21(DE3)pLysS and purified using His-Bind resin; functionality was assessed in comparison with the recombinant wild type by a standard enzyme assay, and growth complementation and kinetic parameters were evaluated. All Asp31 substitutions affected enzyme function; D31E, D31Q and D31N reduced activity by 80,90%, and D31A and D31L by >,90%. All D31 mutants had modified kinetics, ranging from three-fold (D31N) to 283-fold (D31L) increases in Km for dihydrofolate, and 12-fold (D31N) to 223 077-fold (D31L) decreases in kcat/Km. Of the Leu32 substitutions, only L32D caused reduced enzyme activity (67%) and kinetic differences from the wild type (seven-fold increase in Km; 21-fold decrease in kcat/Km). Only minor variations in the Km for NADPH were observed for all substitutions. Whereas the L32F mutant retained similar trimethoprim affinity as the wild type, the L32A mutation resulted in a 12-fold decrease in affinity and the L32D mutation resulted in a seven-fold increase in affinity for trimethoprim. These findings support the hypotheses that Asp31 plays a functional role in binding of the substrate and Leu32 plays a functional role in binding of trimethoprim. [source] Antioxidant Protection Mechanisms And Arachidonic Acid Synthesis Are Altered In Schwann Cells Grown In Elevated GlucoseJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 3 2000C Miinea Accumulating evidence points to oxidative stress as an important factor in the onset of diabetic neuropathy. We have investigated the status of antioxidant protection mechanisms in immortalized rat Schwann cells cultured in high (30 and 50 mM) concentrations of glucose. As compared to growth in 5 mM glucose, the cells contained 40% less reduced glutathione (n =8, p < 0.01). Total superoxide dismutase activity was diminished by more than 50% (n=3; p < 0.001), whereas catalase activity was unchanged. The cellular NADH/NAD+ ratio was progressively increased with increasing medium glucose concentrations. Our previous findings have established that upon exposure of cultured cells to elevated glucose, the proportions of arachidonic acid-containing molecular species (ACMS) in phospholipids are decreased in a pattern similar to alterations exhibited by diabetic nerve. To examine whether biosynthesis of arachidonic acid might be perturbed, confluent cells maintained in either high or low glucose were incubated with either [14C]linoleic acid (18:2) or [14C]dihomo-,-linolenic acid (20:3) and radioactivity incorporated into molecular species of major phospholipid classes was measured. The incorporation of 18:2 either as unchanged fatty acid or into ACMS did not differ as a function of glucose concentration. Negligible labeled 18:3 or 20:3 molecular species were detected. In contrast, the uptake of 20:3 into 18:1/20:4 and 16:0/20:4 phosphatidylcholine and 18:1/20:4 phosphatidylethanolamine, but not into 20:3-containing molecular species, was significantly reduced in cells cultured in 30 mM glucose. These data imply that ,5 desaturase activity is decreased in cells exposed to elevated glucose. This reduced enzyme activity could adversely affect polyunsaturated fatty acid metabolism and might arise as a consequence of impaired scavenging of reactive oxygen species. (Supported by NIH grant DK30577) [source] Methylenetetrahydrofolate reductase polymorphism in Kawasaki diseasePEDIATRICS INTERNATIONAL, Issue 3 2000Hirokazu Tsukahara Abstract Background: A genetic aberration in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene (677 C to T substitution) has been shown to result in reduced enzyme activity. The hypothesis tested in the present study was that a higher proportion of Kawasaki disease (KD) patients with coronary artery lesions (CAL) would have the T677 allele compared with patients without CAL and healthy subjects. Methods: Genotypes for MTHFR were determined in 75 KD patients (male : female ratio 52:23) and 238 healthy subjects (male : female ratio, 110:128) by the polymerase chain reaction and restriction fragment length polymorphism method. Results: The results indicated that female KD patients had a significantly higher frequency of the TT genotype compared with female control subjects. In the female population, the frequency of the TT genotype in patients with initial coronary aneurysm was significantly lower than in patients without this manifestation. Analysis of the data for the male population showed that the frequency of the TT genotype in KD patients developing coronary stenosis, occlusion or myocardial infarction was higher than that in those without these manifestations, although the difference was statistically insignificant. Conclusions: The TT genotype may protect female KD patients against initial aneurysm formation and predispose male KD patients to severe coronary complications. Further large-scale studies may be required to confirm the contribution of homocysteine in the coronary sequelae of KD. [source] Glutathione- S -transferase pi as a model protein for the characterisation of chemically reactive metabolitesPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 2 2008Rosalind E. Jenkins Dr. Abstract Chemically reactive metabolites (CRMs) are thought to be responsible for a number of adverse drug reactions through modification of critical proteins. Methods that defined the chemistry of protein modification at an early stage would provide invaluable tools for drug safety assessment. Here, human GST pi (GSTP) was exploited as a model target protein to determine the chemical, biochemical and functional consequences of exposure to the hepatotoxic CRM of paracetamol (APAP), N -acetyl- p -benzoquinoneimine (NAPQI). Site-specific, dose-dependent modification of Cys47 in native and His-tagged GSTP was revealed by MS, and correlated with inhibition of glutathione (GSH) conjugating activity. In addition, the adaptation of iTRAQ labelling technology to define precisely the quantitative relationship between covalent modification and protein function is described. Multiple reaction monitoring (MRM)-MS of GSTP allowed high sensitivity detection of modified peptides at physiological levels of exposure. Finally, a bioengineered mutant cytochrome P450 with a broad spectrum of substrate specificities was used in an in vitro reaction system to bioactivate APAP: in this model, GSTP trapped the CRM and exhibited both reduced enzyme activity and site-specific modification of the protein. These studies provide the foundation for the development of novel test systems to predict the toxicological potential of CRMs produced by new therapeutic agents. [source] | |||||||||||||