Properties and role of an internal aldimine lysine of pyridoxal phosphate enzymes
The cytoplasmic isozyme of aspartate aminotransferase had been reductively methylated with formaldehyde and NaCNBH/sub 3/ at pH 6.0. Peptide fractionation of the tryptic digests of the methylated enzyme reveals only two modified amino groups, one in each of two peptides. Phenylisothiocyanate derivatization and subsequent chromatography was used to separate mono- and dimethylated lysines. The active site lysine-258 was at least 87% dimethylated. The apoenzyme methylated at the active site lysine-258 accepts the coenzyme pyridoxal phosphate (PLP) at is active site at a ratio of 1 mole of coenzyme/mole monomer as seen by circular dichroism (CD) as does native enzyme. The visible CD spectrum indicates the PLP coenzyme is held in a different environment than that of the native enzyme. The absorption spectrum exhibits maxima different from the native enzyme and are maxima corresponding to coenzyme bound to the active site without Schiff's base formation. The CD spectrum in the far UV indicates no major changes in secondary structure with reductive methylation. The active site methylated holoenzyme can carry out half transamination reactions and forms spectroscopically detectable enzyme-substrate complexes with amino and keto acids that form and interconvert at a slower rate than in native enzyme. Apoenzyme reductively methylated with (/sup 13/C)-formaldehyde exhibits a resonance unique for that of the active site lysine-258 in the /sup 13/C) NMR spectrum.
- Research Organization:
- Virginia Commonwealth Univ., Richmond (USA)
- OSTI ID:
- 6439848
- Resource Relation:
- Other Information: Thesis (Ph. D)
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
AMINOTRANSFERASES
CHEMICAL PROPERTIES
FRACTIONATION
LYSINE
METHYLATION
CARBON 13
CHROMATOGRAPHY
FORMALDEHYDE
NMR SPECTRA
NUCLEAR MAGNETIC RESONANCE
ALDEHYDES
AMINO ACIDS
CARBON ISOTOPES
CARBOXYLIC ACIDS
CHEMICAL REACTIONS
ENZYMES
EVEN-ODD NUCLEI
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
NITROGEN TRANSFERASES
NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
RESONANCE
SEPARATION PROCESSES
SPECTRA
STABLE ISOTOPES
TRANSFERASES
550200* - Biochemistry