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

[pyruvate dehydrogenase deficiency]

Human pyruvate dehydrogenase (E 1 ), a heterotetramer (alpha 2 beta 2 ), is the first component of the pyruvate dehydrogenase complex (PDC ). E 1 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 E 1 -deficient patients , M 181 V , R 349 H , and P 188 L (P 188 A mutant E 1 was used because of the very low level of expression of P 188 L ), to investigate the functional roles of these three amino acid residues . P 188 A mutant E 1 was much less thermostable than the wild-type E 1 . The kcats of M 181 V and P 188 A mutant E 1 s determined in the PDC reaction were 38 and 24 % of that of the wild-type enzyme , respectively . The apparent Km for TPP for M 181 V 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 , P 188 A had similar Kms for the coenzyme and the substrate as the wild-type . Km values for R 349 H were not determined due to the extremely low activity of this mutant (1 .2 % of the wild-type E 1 -specific activity measured in the PDC assay ). Wild-type E 1 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 E 1 . M 181 V required a higher concentration of ThTTPP for inactivation than the wild-type and P 188 A E 1 s . The results of circular dichroism spectropolarimetry in the far UV region indicated that there were no major changes in the secondary structure of M 181 V , P 188 A , and R 349 H E 1 s . 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 E 1 's activity , methionine-181 is involved in the binding of TPP , and proline-188 is necessary for structural integrity of E 1 .

Diseases
Validation

Diseases presenting "very low level" symptom

cadasil

carcinoma of the gallbladder

cowden syndrome

proteus syndrome

pyruvate dehydrogenase deficiency

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