Detection of a gem-diamine and a stable quinonoid intermediate in the reaction catalyzed by serine-glyoxylate aminotransferase from Hyphomicrobium methylovorum.
BACKGROUND
The enzyme L-serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a PLP-containing enzyme that catalyzes the conversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The cloned enzyme expressed in Escherichia coli is isolated as a mixture of the E:PLP and E:PMP forms. The PLP form of the enzyme has a maximum absorbance at 413 nm.


METHODS
Uv-visible spectra of SGAT were obtained using an HP-8453 diode array spectrophotometer in the absence and presence of substrates and substrate analogs. Pre-steady state kinetic studies were carried out using an OLIS rapid scanning spectrophotometer in the rapid scanning mode.


RESULTS
Incubation of the enzyme with a saturating concentration D-serine leads to a shift in the 413 nm peak to 421 nm that is ascribed to the external aldimine. The reverse stereochemistry of D-serine does not allow for abstraction of the C alpha proton by the epsilon-amine of the active site lysine residue leading to an abortive external aldimine intermediate. Pre-steady state studies pushing SGAT against D-serine leads to a rapid decrease in the 413 nm peak and an increase at approximately 330 nm with an associated rate constant of 47 s(-1) at pH 7.6. This is followed by a slower decrease (0.26 s(-1)) at 330 nm and an increase and shift of the 413 nm peak to 421 nm. The intermediate species that absorbs at approximately 330 nm is attributed to the gem-diamine intermediate. The rate of the fast phase increases with pH and increase in rate is likely due to the deprotonation of an enzymatic group that accepts a proton from the alpha-amine of D-serine. In the presence of hydroxypyruvate and ammonia the enzyme spectra display an increase in absorbance at 521 nm that occurs on the order of minutes. The shape and position of the 521 nm species is consistent with a quinonoid intermediate.


CONCLUSIONS
The data suggest a non-enzymatic reaction between hydroxypyruvate and ammonia to form an imine which will be in equilibrium with the enamine. A mechanism is proposed by which the enamine reacts with the PLP form of SGAT to generate the stable highly conjugated quinonoid intermediate.
Karsten
William E
WE
Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, OK 73019, USA.
Cook
Paul F
PF
D003959:Diamines / Q000737:chemistry*; D020582:Hyphomicrobium / Q000201:enzymology*; D015394:Molecular Structure; D011773:Pyruvates / Q000737:chemistry / Q000378:metabolism; D011809:Quinones / Q000737:chemistry*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D000637:Transaminases / Q000737:chemistry / Q000378:metabolism*
D003959:Diamines; D011773:Pyruvates; D011809:Quinones; D012694:Serine; C012375:hydroxypyruvic acid; D000637:Transaminases; C022636:serine-glyoxylate aminotransferase