Reaction mechanism of Escherichia coli cystathionine gamma-synthase: direct evidence for a pyridoxamine derivative of vinylglyoxylate as a key intermediate in pyridoxal phosphate dependent gamma-elimination and gamma-replacement reactions

Biochemistry. 1990 Jan 16;29(2):442-51. doi: 10.1021/bi00454a020.

Abstract

Cystathionine gamma-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-L-homoserine (OSHS) and L-cysteine via a gamma-replacement reaction. In the absence of L-cysteine, OSHS undergoes an enzyme-catalyzed, gamma-elimination reaction to form succinate, alpha-ketobutyrate, and ammonia. Since elimination of the gamma-substituent is necessary for both reactions, it is reasonable to assume that the replacement and elimination reaction pathways diverge from a common intermediate. Previously, this partitioning intermediate has been assigned to a highly conjugated alpha-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the gamma-replacement and gamma-elimination reactions of cystathionine gamma-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The gamma-elimination reaction is characterized by a rapid decrease in the amplitude of the enzyme internal aldimine spectral band at 422 nm with a concomitant appearance of a new species which absorbs in the 300-nm region. A 485-nm species subsequently accumulates in a much slower relaxation. The gamma-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (approximately 3 ms). This relaxation is followed by a decrease in absorbance at 425 nm that is tightly coupled to the appearance of a species which absorbs in the 300-nm region. Reaction of the substrate analogues L-alanine and L-allylglycine with cystathionine gamma-synthase results in bleaching of the 422-nm absorbance and the appearance of a 300-nm species. In the absence of L-cysteine, L-allylglycine undergoes facile proton exchange; in the presence of L-cysteine, L-allylglycine undergoes a gamma-replacement reaction to form a new amino acid, gamma-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an alpha-imino beta,gamma-unsaturated pyridoxamine derivative with lambda max congruent to 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway.

Publication types

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

MeSH terms

  • Alanine / metabolism
  • Allylglycine / metabolism
  • Carbon-Oxygen Lyases*
  • Cysteine / metabolism
  • Escherichia coli / enzymology*
  • Fatty Acids, Monounsaturated / metabolism*
  • Homoserine / analogs & derivatives
  • Homoserine / metabolism
  • Kinetics
  • Lyases / metabolism*
  • Molecular Structure
  • Pyridoxal Phosphate / pharmacology*
  • Pyridoxamine / metabolism*
  • Spectrophotometry
  • Spectrophotometry, Ultraviolet

Substances

  • Fatty Acids, Monounsaturated
  • Allylglycine
  • O-succinylhomoserine
  • vinylglyoxylate
  • Pyridoxal Phosphate
  • Pyridoxamine
  • Homoserine
  • O-succinylhomoserine (thiol)-lyase
  • Lyases
  • Carbon-Oxygen Lyases
  • Cysteine
  • Alanine