NMR STUDY OF ASPARTATE AMINOTRANSFERASE

1989 Fiscal Year Final Research Report Summary

• Project/Area Number: 63570120
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: General medical chemistry
• Research Institution: KUMAMOTO UNIVERSITY
• Principal Investigator: HIGAKI Tsuyoshi KUMAMOTO UNIV. COLLEGE OF MED. SCI. RESEARCHER, 医療技術短期大学部, 助手 (70128304)
• Co-Investigator(Kenkyū-buntansha): TANASE Sumio KUMAMOTO UNIV. MEDICAL SCHOOL LECTURER, 医学部, 講師 (20112401)
• Project Period (FY): 1988 – 1989
• Keywords: Aspartate Aminotransferase (AspAT) / Nuclear Magnetic Resonance (NMR) / Stable Isotope / 安定同位体

Research Abstract

Aspartate aminotransferase (AspAT, EC 2.6.1.1., MW - 93,000) is composed of two identical subunits. The minimal mechanism of AspAT, as deduced by the enormous wealth of model, spectroscopic, and X-ray crystallographic studies, is shown to proceed through a double replacement mechanism, that is the pyridoxal-P coenzyme shuttling between the pyridoxal and pyridoxamine forms. Thus, it is expected that C4' of the coenzyme, which has been shown to form the internal aldimine bond with Lys-258 of the enzyme, would serve as a sensitive chemical shift probe for the ionization states of both the imine nitrogen and its microenvironment. The present paper reports on the ^<13>C NMR spectroscopy of the porcine cytosolic isoenzyme of AspAT reconstituted with [4'-^<13>C] pyridoxal-P.
1. The chemical shift of C4' of the internal aldimine in the absence of ligands was determined to be 165.3 ppm at pH 4.8 and was found to undergo a 5 ppm shift to a higher field with increasing pH, with a pK value of 6.3.
2. The resonance line for C4' of the coenzyme at pH 7.5 was found to shift downfield (by about 4 ppm) upon the addition of a saturating concentration of succinate which is a competitive inhibitor. The effect of succinate on the C4' chemical shift was examined further at various pH values and the titration curve thus obtained yielded a pK value of 7.7.
3. Reaction with cysteinesulfinate caused complete disappearance of the aldimine signal at 165 ppm with a concomitant appearance of a new signal at 39.6 ppm.
4.2-Methylaspartate reacts with the pyridoxal form of AspAT to form a stable external aldimine complex, since the substitution of the methyl group at position 2 of the amino acid precludes further reaction leading to transamination. The resonance line of C4' did not display any pH dependent chemical shift effects between the pH range of 5 - 9 in a saturating concentration of this substrate analog.