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Characterization of a missense mutation at histidine-44 in a pyruvate dehydrogenase-deficient patient
Scott J Jacobia1, Lioubov G Korotchkina, Mulchand S Patel
1Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.
Insights
Genetic defects in pyruvate dehydrogenase complex (PDC) cause severe health issues. A specific E1alpha mutation (alphaH44R) impairs thiamine pyrophosphate (TPP) binding, impacting enzyme function and potentially explaining patient symptoms.
Area of Science:
- Biochemistry
- Genetics
- Enzymology
Background:
- Pyruvate dehydrogenase complex (PDC) genetic defects lead to lactic acidosis and neurological problems.
- Mutations in the E1alpha subunit are common causes of PDC deficiency.
Purpose of the Study:
- To kinetically characterize the alphaH44R missense mutation in human E1alpha.
- To understand the impact of substitutions at histidine-15 on enzyme kinetics and thiamine pyrophosphate (TPP) binding.
Main Methods:
- Created and purified three recombinant human E1 proteins: alphaH44R, alphaH44Q, and alphaH44A.
- Performed kinetic characterization, measuring V(max) and K(m) for TPP.
- Assessed phosphorylation and dephosphorylation rates.
Main Results:
- The alphaH44R mutation significantly decreased V(max) (to 6% of wild-type) and increased TPP K(m) (3-fold).
- Larger side chain substitutions at residue 15 further disrupted TPP binding and enzyme activity.
- Phosphorylation and dephosphorylation rates were similar to wild-type, indicating these steps were not primarily affected.
Conclusions:
- The volume of the residue at site 15 is crucial for TPP binding in E1alpha.
- The alphaH44R mutation disrupts E1alpha active site function, providing a biochemical basis for patient symptoms.
- Enzyme kinetics reveal specific mechanisms underlying PDC deficiency caused by E1alpha mutations.
Abstract:
Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.