Part A. The pyruvate dehydrogenase complex. Access the pyruvate dehydrogenase E1
ID: 63319 • Letter: P
Question
Part A. The pyruvate dehydrogenase complex. Access the pyruvate dehydrogenase E1 component subunit alpha, mitochondrial: http://www.uniprot.org/uniprot/P08559. The purpose of this problem set is for you to work with and present on-line data.
1. What does the transit peptide do? Provide the FASTA-formatted transit peptide sequence for P08559 in Courier font, Size 12. The primary amino acid sequence can be found by clicking on the FASTA tab under Sequences, and you can copy and paste to obtain the sequence.
2. What KNOWN postranslational modifications are there in the protein? List the type and location.
3. What is the estimated pI and molecular weight of the active protein?
4. What are the enzymes PDK1-4 and what effect do they have on pyruvate dehydrogenase E1 subunit
alpha?
5. Review the natural variants. What is PDHAD and why do individuals with this disease suffer from
lactic acidosis (high levels of lactic acid in the bloodstream)? Use a biochemical explanation.
Part B. The TCA Cycle
Please use your own words in providing your answers. Citations should be provided.
Sodium fluoroacetate was used as a pesticide for a number of years and is highly toxic to all mammals. Provide a brief history of the compound. What is the proposed mechanism of inhibition and how does this inhibition lead to issues with glycolysis? A reference article for the mechanistic aspects is below: http://www.pnas.org/content/93/24/13699.abstract
Explanation / Answer
Since this question has a lot of sub-parts, I will answer the first four according to the answering guidelines.
1) A transit peptide is basically a signal sequence which is present at the N-terminus of newly synthesized proteins and is responsible for directing the protein to a specialized destination via classical secretory pathway. The destination may include organelle such as mitochondria and chloroplast.
Sequqnce of transit peptide in Fasta format:
>sp|P08559|1-30
MRKMLAAVSRVLSGASQKPASRVLVASRNF
2) Mainly phosphorylation, succinylation and acetylation are known PTMs in this protein. Following are the sites marked with the position of amino acids in the sequence.
Acetylation: 63, 244, 313, 321, 336
Succinylation: 63, 244, 277, 313, 385
Phosphorylation: 231, 232, 293, 295, 300, 301
3) The estimated pI would be 6.51 and the molecular weight of the active protein would be 40 kDa.
4) PDK stands for Pyruvate dehydrogenase kinase. These enzymes inactivate pyruvate dehydrogenase by phosphorylating it using ATP.