Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine

Biochemistry. 1997 Feb 18;36(7):1730-9. doi: 10.1021/bi962264x.

Abstract

Dihydrodipicolinate synthase (DHDPS) catalyzes the formation of dihydrodipicolinate from pyruvate and L-aspartate beta-semialdehyde (ASA). A parallel initial velocity pattern that displays competitive substrate inhibition by ASA and dead-end inhibition patterns obtained at pH 8 are consistent with a ping pong kinetic mechanism for DHDPS. The results suggest that pyruvate binds to free enzyme with subsequent formation of a Schiff base with an enzymic lysine residue followed by binding of ASA to the F enzyme form to initiate the second half-reaction. At low pH (5.7) the initial velocity and dead-end inhibition patterns are consistent with a sequential steady state ordered kinetic mechanism with pyruvate binding to enzyme prior to ASA. The irreversible step in the reaction, leading to the ping pong kinetic mechanism at high pH, is proposed to be loss of a proton from the methyl group of pyruvate in Schiff base with enzyme to form an enamine intermediate. Consistent with this proposal is the change to a sequential steady state ordered kinetic mechanism at low pH at or below the pK of the enamine intermediate. L-Lysine is an allosteric inhibitor of the DHDPS reaction that causes partial inhibition (approximately 90%) at saturating concentrations. Inhibition patterns for L-lysine vs pyruvate and ASA suggest that lysine binds to the F enzyme form at pH 8 with a Ki value of about 0.3 mM. An examination of the effects of different L-lysine concentrations on the kinetic parameters V/Kpyruvate, V/KASA, and V indicate that L-lysine decreases only the values of V/KASA and Vmax, which is consistent with the inhibitory effects of lysine manifested on the second half-reaction. In contrast at low pH the data suggest L-lysine binds to free enzyme with an inhibition constant of about 5 mM.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Allosteric Regulation
  • Aspartic Acid / analogs & derivatives
  • Aspartic Acid / pharmacology
  • Binding, Competitive
  • Escherichia coli / enzymology*
  • Escherichia coli / metabolism
  • Hydro-Lyases / antagonists & inhibitors*
  • Hydro-Lyases / isolation & purification
  • Hydro-Lyases / metabolism*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Lysine / pharmacology*
  • Substrate Specificity / drug effects

Substances

  • Aspartic Acid
  • aspartic semialdehyde
  • Hydro-Lyases
  • 4-hydroxy-tetrahydrodipicolinate synthase
  • Lysine