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Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat
1Department of Biochemistry, Hamamatsu University School of Medicine, Shizuoka, Japan.
Abstract:
The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45,834 Da. Comparison of the N-terminal amino acid sequence of mitochondrial serine:pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43,210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N-terminal extension peptide (pre-sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative alpha-helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine:pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine:pyruvate aminotransferase.
Insights
Researchers determined the rat liver mitochondrial serine:pyruvate aminotransferase precursor mRNA sequence. This revealed a 24-amino acid pre-sequence essential for mitochondrial import and identified a potential active site lysine residue.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Mitochondrial serine:pyruvate aminotransferase is crucial for amino acid metabolism.
- Understanding its precursor and import mechanism is vital for cellular function.
Purpose of the Study:
- To determine the nucleotide sequence of the mRNA encoding rat liver mitochondrial serine:pyruvate aminotransferase precursor.
- To characterize the pre-sequence responsible for mitochondrial import.
- To predict the enzyme's active site.
Main Methods:
- Nucleotide sequencing of cDNA clones.
- N-terminal amino acid sequencing.
- Bioinformatic analysis (helical wheel, secondary structure prediction).
Main Results:
- The mRNA sequence (≥1533 nucleotides) encodes a 414-amino acid precursor.
- The mature enzyme comprises 390 amino acids.
- A 24-amino acid N-terminal pre-sequence with amphipathic alpha-helical properties was identified.
- Predicted secondary structures suggest a conserved feature among related aminotransferases.
Conclusions:
- The pre-sequence facilitates specific mitochondrial import.
- A lysine residue binding pyridoxal phosphate is proposed as the active site of serine:pyruvate aminotransferase.
- The findings provide insights into enzyme structure-function relationships and mitochondrial targeting.