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 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.
Brzović
P
P
Department of Biochemistry, University of California, Riverside 92521.
Holbrook
E L
EL
Greene
R C
RC
Dunn
M F
MF
D000409:Alanine / Q000378:metabolism; D000501:Allylglycine / Q000378:metabolism; D019757:Carbon-Oxygen Lyases; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D005229:Fatty Acids, Monounsaturated / Q000378:metabolism*; D006714:Homoserine / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008190:Lyases / Q000378:metabolism*; D015394:Molecular Structure; D011732:Pyridoxal Phosphate / Q000494:pharmacology*; D011733:Pyridoxamine / Q000378:metabolism*; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D005229:Fatty Acids, Monounsaturated; D000501:Allylglycine; C014196:O-succinylhomoserine; C059218:vinylglyoxylate; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D006714:Homoserine; C022696:O-succinylhomoserine (thiol)-lyase; D008190:Lyases; D019757:Carbon-Oxygen Lyases; D003545:Cysteine; D000409:Alanine