콘텐츠 본문
논문 국내 국제전문학술지(SCI급) Domain Closure in the Catalytic Chains of E. coli Aspartate Transcarbamolyse Influences the Kinetic Mechanism
- 학술지 구분 국제전문학술지(SCI급)
- 게재년월 1995-01
- 저자명 이봉호단독
- 학술지명 J. Biol. Chem.
- 발행국가 국내
- 논문언어 한국어
논문 초록 (Abstract)
The closure of the two domains of the catalytic chains of Escherichia coli aspartate transcarbamoylase, which is critical for completion of the T → R transition, is stabilized by salt-bridges between Glu-50 and both Arg-167 and Arg-234. Mutation of Glu-50 to Ala shifts the enzyme toward a low activity, low affinity state (Newton, C. J., and Kantrowitz, E. R.(1990) Biochemistry, 29, 1444-1451). Kinetic isotope effects (KIE) and equilibrium isotope exchange kinetics (EIEK) have been used to probe the dynamic properties of the Glu-50 → Ala enzyme. Unlike the behavior of the wild-type enzyme, the observed kinetic isotope effect for 13C versus12C at the carbonyl group of carbamoyl phosphate (CP) increased upon the binding of ligands which promote the formation of the R-state (Asp, N-phosphonacetyl-L-aspartate (PALA), or ATP). The maximum rate for the [14C]Asp ⇋ Carbamoyl aspartate (CAsp) exchange with the Glu-50 → Ala enzyme was 500-fold slower than for the wild-type enzyme; however, the rate for the [14C]CP ⇋ CAsp exchange was only 50-fold slower, reversing the relative rates observed with the wild-type enzyme. In addition, upon variation of substrate pairs involving Asp or CAsp, loss of inhibition effects in the CP ⇋ CAsp exchange indicated that the Glu-50 → Ala substitution caused the kinetic mechanism for the mutant enzyme to shift from ordered to random. Computer simulations of the EIEK data indicate that the Glu-50 → Ala mutation specifically causes strong decreases in the rates of catalysis and association-dissociation for Asp and CAsp, with minimal effects on the CP and Pi on-off rates. With substrates bound, the Glu-50 → Ala enzyme apparently does not attain a full R-state conformation. The PALA-activated Glu-50 → Ala enzyme, however, exhibits substrate affinities comparable to those for the wild-type enzyme, but fails to restore the preferred order substrate binding. Unlike the wild-type enzyme, both the T and R-states of the Glu-50 → Ala enzyme contribute to catalysis. A third state, I, is proposed for the Glu-50 → Ala enzyme, in which random order substrate binding is exhibited, and the catalytic step contributes significantly to overall rate limitation.
