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A Tripatara

Publications and source records attributed to A Tripatara.

5 recordsLinked to original sources
PubMed1999

Characterization of point mutations in patients with pyruvate dehydrogenase deficiency: role of methionine-181, proline-188, and arginine-349 in the alpha subunit.

Human pyruvate dehydrogenase (E1), a heterotetramer (alpha2beta2), is the first component of the pyruvate dehydrogenase complex (PDC). E1 catalyzes the thiamin pyrophosphate (TPP)-dependent decarboxylation of pyruvate and the reductive acetylation of the dihydrolipoamide acetyltransferase component. Site-directed mutagenesis was employed to recreate three point mutations in the alpha subunit identified in E1-deficient patients, M181V, R349H, and P188L (P188A mutant E1 was used because of the very low level of expression of P188L), to investigate the functional roles of these three amino acid residues. P188A mutant E1 was much less thermostable than the wild-type E1. The kcats of M181V and P188A mutant E1s determined in the PDC reaction were 38 and 24% of that of the wild-type enzyme, respectively. The apparent Km for TPP for M181V increased significantly (approx 250-fold when determined in the PDC assay), while the apparent Km for pyruvate increased by only about 3-fold. In contrast, P188A had similar Kms for the coenzyme and the substrate as the wild-type. Km values for R349H were not determined due to the extremely low activity of this mutant (1.2% of the wild-type E1-specific activity measured in the PDC assay). Wild-type E1 displayed a lag phase in the progress curve of the PDC reaction measured in the presence of low TPP concentrations (below 1 microM) only. All mutants had a lag phase that was not eliminated even at very high TPP concentrations, suggesting modifications in the conformation of the active site. Kinetic analysis indicated thiamin 2-thiothiazolone pyrophosphate (ThTTPP) to be an intermediate analog for wild-type human E1. M181V required a higher concentration of ThTTPP for inactivation than the wild-type and P188A E1s. The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M181V, P188A, and R349H E1s. These mutant enzymes exhibited negative dichroic spectra at about 330 nm only in the presence of high TPP concentrations. This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.

2,6-Dichloroindophenol↗
PubMed1996

Three new mutations of the pyruvate dehydrogenase alpha subunit: a point mutation (M181V), 3 bp deletion (-R282), and 16 bp insertion/frameshift (K358SVS-->TVDQS).

Three novel mutations in the coding region of E1 alpha gene were found in three PDC-deficient male patients, including a missense mutation (M181V), a 3 bp deletion (AGA, corresponding to R282), and a 16 bp insertion (CAGTGGATCAAGTTTA), causing a frameshift starting with lysine 358 and resulting in decrease of both E1 subunits.

Cells, Cultured↗
PubMed1993

A new start site for Escherichia coli RNA polymerase at an engineered short region of non-complementarity in double-stranded DNA.

We have constructed versions of the bacteriophage PRM promoter containing short (9 or 12 base pairs) regions of DNA mismatches ("bubble") which include the authentic transcription start site of the unmodified promoter. These constructs direct transcription initiation at positions near the genuine PRM start site. In addition a new start site (designated Pbub) is observed in the region of non-complementarity, from which RNA synthesis proceeds in the opposite direction. The ability to initiate the divergent transcripts is specific to holo enzyme. Mapping of the Pbub start sites shows that they are but a few base pairs upstream of the edge of the bubble. Thus, with respect to the single-stranded region, the location of the start site is no different for Pbub than it is for open complexes at promoters. Compared with an unmodified PRM promoter, the region protected by RNA polymerase from digestion by DNase I is extended in the downstream direction (with respect to the PRM start) at the promoters bearing mismatches; this is consistent with the binding of the divergently transcribing RNA polymerase. Interestingly, cI protein represses rather than activates RNA synthesis originating in the PRM direction, indicating yet another aspect in which the complexes formed at these constructs differ from open complexes at the unmodified promoter.

Bacteriophage lambda↗
PubMed1993

Interference by PR-bound RNA polymerase with PRM function in vitro. Modulation by the bacteriophage lambda cI protein.

Activation of the weak PRM promoter by cI protein is an essential process in the establishment of lysogeny. Much evidence has accumulated that cI protein binds cooperatively to the operators OR1 and OR2 and that protein at the OR2 site contacts RNA polymerase to facilitate open complex formation at the PRM promoter. We had shown previously in vitro that RNA polymerase situated at the nearby PR promoter could interfere with open complex formation at PRM and that an additional mechanism of PRM activation in vitro involved cI-mediated RNA polymerase exclusion from PR. Here we further characterize this second indirect mode of activation. We demonstrate the addition of cI and inactivation of the PR promoter activate open complex formation at PRM similarly over the temperature range from 37 to 20 degrees C in which the extent of activation decreases from 8- to 2-fold. We also show that the binding of cI protein to OR1 is sufficient to effect an increase in the rate of synthesis of abortive RNA products at PRM. This result is difficult to explain based on direct cI-RNA polymerase contacts alone but is readily interpreted in terms of our previously proposed model involving the exclusion of an interfering RNA polymerase from binding at PR.

Bacteriophage lambda↗