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Vanadate catalyzes photocleavage of adenylate kinase at proline-17 in the phosphate-binding loop
C R Cremo1, J A Loo, C G Edmonds
1Biochemistry and Biophysics Department, Washington State University, Pullman 99164-4660.
Abstract:
Irradiation of adenylate kinase (AK) from chicken muscle with 300-400-nm light in the presence of 0.25 mM vanadate ion first inactivated the enzyme and then cleaved the polypeptide chain near the NH2 terminus. The addition of the multisubstrate analogue, P1,P5-bis(5'-adenosyl) pentaphosphate, prevented both effects. ATP, but not AMP, blocked both inactivation and cleavage in a saturable manner, suggesting that both effects were due to modification at the ATP-binding site. The polypeptide products of the photocleavage were isolated by HPLC and characterized by amino acid composition, peptide sequencing, and mass spectral analyses. The predominant (greater than 90%) small peptide fragment contained the first 16 amino acids from the amino terminus of the enzyme. The amino terminus of this peptide contained an acetylated serine, and the "carboxy" terminus was modified by a cyclized gamma-aminobutyric acid which originated from photooxidation and decarboxylation of proline-17 by vanadate. Edman sequencing indicated that the majority of the large peptide fragment (Mr approximately 19,500) was amino-terminal blocked, but a small portion was sequenceable starting at either glycine-18 (7%) or serine-19 (2%). These studies indicate that in the ATP-AK complex proline-17 is close to the phosphate chain of ATP but not AMP, consistent with the latest evaluation of nucleotide-binding sites on mitochondrial matrix AK by X-ray crystallography [Diederichs, K., & Schulz, G.E. (1991) J. Mol. Biol. 217, 541-549]. Furthermore, this is the first report that an amino acid other than serine can be involved in vanadate-promoted photocleavage reactions.
Insights
Vanadate-promoted photocleavage of adenylate kinase (AK) inactivated the enzyme and cleaved its polypeptide chain near the N-terminus. This reaction, occurring at the ATP-binding site, was prevented by ATP and a multisubstrate analogue.
Area of Science:
- Biochemistry
- Enzymology
- Photochemistry
Background:
- Adenylate kinase (AK) plays a crucial role in cellular energy metabolism.
- Understanding the structural and functional aspects of AK, particularly its nucleotide-binding sites, is important for biochemical research.
Purpose of the Study:
- To investigate the mechanism of vanadate-promoted photocleavage of chicken muscle adenylate kinase.
- To identify the site of cleavage and characterize the resulting peptide fragments.
- To elucidate the role of ATP binding in the photocleavage reaction.
Main Methods:
- Irradiation of adenylate kinase with 300-400-nm light in the presence of vanadate.
- Use of multisubstrate analogue P1,P5-bis(5 omino) pentaphosphate and ATP/AMP to study protection mechanisms.
- Isolation and characterization of peptide fragments using HPLC, amino acid composition, peptide sequencing, and mass spectrometry.
Main Results:
- Photocleavage inactivated adenylate kinase and cleaved the polypeptide near the N-terminus.
- ATP, but not AMP, protected against inactivation and cleavage, indicating modification at the ATP-binding site.
- The predominant peptide fragment (1-16 amino acids) had an acetylated N-terminus and a modified C-terminus (cyclized gamma-aminobutyric acid from proline-17).
- Proline-17 is located near the ATP phosphate chain in the ATP-AK complex, consistent with crystallographic data.
Conclusions:
- Vanadate-promoted photocleavage is a useful tool for studying enzyme structure and function.
- The results provide insights into the ATP-binding site of adenylate kinase.
- This study reports a novel instance of vanadate-promoted photocleavage involving an amino acid other than serine.