Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae

Biochemistry
Ashwani Kumar VashishthaPaul F Cook

Abstract

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA a...Continue Reading

References

Aug 1, 1989·The Biochemical Journal·N D Priestley, J A Robinson
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Sep 1, 2006·Cell Biochemistry and Biophysics·Hengyu XuPaul F Cook

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Citations

Sep 8, 2015·Archives of Biochemistry and Biophysics·Ashwani K VashishthaPaul F Cook
Aug 21, 2020·MSystems·Jacqueline R Morey, Thomas E Kehl-Fie
May 20, 2009·Biochemistry·Ashwani Kumar VashishthaPaul F Cook

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