Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli

Biochemistry. 1992 Jan 28;31(3):799-805. doi: 10.1021/bi00118a023.


The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aspartate Kinase / antagonists & inhibitors
  • Aspartate Kinase / metabolism*
  • Aspartic Acid / analogs & derivatives
  • Aspartic Acid / metabolism
  • Aspartic Acid / pharmacology
  • Binding Sites
  • Escherichia coli / enzymology*
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Phosphorus
  • Substrate Specificity


  • Phosphorus
  • Aspartic Acid
  • Aspartate Kinase