Porcine cytosolic aspartate aminotransferase reconstituted with (4 prime - sup 13 C)pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by sup 13 C NMR spectroscopy

Journal Article · Tue Mar 05 00:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00223a032· OSTI ID:5715317

Higaki, Tsuyoshi ^[1]; Tanase, Sumio; Nagashima, Fujio; Morino, Yoshimasa ^[2]; Scott, A I; Williams, H J; Stolowich, N J ^[3]

---

1. Kumamoto Univ. College of Medical Science (Japan) Kumamoto Univ. Medical School (Japan)
2. Kumamoto Univ. Medical School (Japan)
3. Texas A and M Univ., College Station (United States)

Apoenzyme samples of aspartate aminotransferase (AspAT) purified from the cytosolic fraction of pig heart were reconstituted with (4{prime}-{sup 13}C)pyridoxal 5{prime}-phosphate (pyridoxal-P). The {sup 13}C NMR spectra of AspAT samples thus generated established the chemical shift of 165.3 ppm for C4{prime} of the coenzyme bound as an internal aldimine with lysine 258 of the enzyme at pH 5. In the absence of ligands the chemical shift of C4{prime} was shown to be pH dependent, shifting 5 ppm upfield to a constant value of 160.2 ppm above pH 8, the resulting pK{sub a} of 6.3 in agreement with spectrophotometric titrations. The addition of the competitive inhibitor succinate to the internal aldimine raises the pK{sub a} of the imine to 7.8, consistent with the theory of charge neutralization in the active site. In the presence of saturating concentrations of 2-methylaspartic acid the C4{prime} signal of the coenzyme was shown to be invariant with pH and located at 162.7 ppm, midway between the observed chemical shifts of the protonated and unprotonated forms of the internal aldimine. Finally, the line widths of the C4{prime} resonance under the various conditions were measured and qualitatively compared. The results are discussed in terms of the current mechanism and molecular models of the active site of AspAT.

• OSTI ID: 5715317
• Journal Information: Biochemistry; (United States), Vol. 30:9; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
AMINOTRANSFERASES
MOLECULAR STRUCTURE
COENZYMES
NUCLEAR MAGNETIC RESONANCE
ASPARTIC ACID
CARBON 13
CHEMICAL SHIFT
CYSTEINE
LIGANDS
PH VALUE
PYRIDOXAL
ALDEHYDES
AMINO ACIDS
AZINES
CARBON ISOTOPES
CARBOXYLIC ACIDS
ENZYMES
EVEN-ODD NUCLEI
HETEROCYCLIC COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
NITROGEN TRANSFERASES
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PROTEINS
PYRIDINES
RESONANCE
STABLE ISOTOPES
THIOLS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques