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

Cystathionine γ-synthase catalyzes a pyridoxal phosphate dependent synthesis of cystathionine from O-succinyl-l-homoserine (OSHS) and l-cysteine via a γ-replacement reaction. In the absence of l-cysteine, OSHS undergoes an enzyme-catalyzed, γ-elimination reaction to form succinate, α-ketobutyrate, and ammonia. Since elimination of the γ-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 α-iminovinylglycine quininoid (Johnston et al., 1979a). The experiments reported herein support an alternative assignment for the partitioning intermediate. We have examined the γ-replacement and γ-elimination reactions of cystathionine γ-synthase via rapid-scanning stopped-flow and single-wavelength stopped-flow UV-visible spectroscopy. The γ-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 γ-replacement reaction shows a red shift of the 422-nm peak to 425 nm which occurs in the experiment dead time (~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 γ-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 γ-replacement reaction to form a new amino acid, γ-methylcystathionine. No long-wavelength-absorbing species accumulate during either of these reactions. These results establish that the partitioning intermediate is an α-imino β,γ-unsaturated pyridoxamine derivative with λmax ≃ 300 nm and that the 485-nm species which accumulates in the elimination reaction is not on the replacement pathway. © 1990, American Chemical Society. All rights reserved.