A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae.

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Research Abstract Details 

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A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Abstract Text:

Hengyu Xu,Susan S Alguindigue,Ann H West,Paul F Cook,

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Publishing Authors By Initials

H Xu,SS Alguindigue,AH West,PF Cook,

For similar enzymes and coenzymes: enzymes: oxidoreductases: oxidoreductases acting on ch-nh group donors: saccharopine dehydrogenases research abstracts see: enzymes and coenzymes: enzymes: oxidoreductases: oxidoreductases acting on ch-nh group donors: saccharopine dehydrogenases research

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A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Biochemistry

VOLUME: 46

Page Numbers: 871-82

Journal Abbreviation: Biochemistry

ISSN: 0006-2960

DAY: 23

MONTH: Jan

YEAR: 2007

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Information

Number of References:

LANGUAGE: eng

NlmUniqueID: 370623

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Keywords Mesh Terms:

KEYWORDS: Saccharopine Dehydrogenases

MESH TERMS: metabolism

Chemical & Substance for Abstract: A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae. Information

Substance Name: Saccharopine Dehydrogenases

Registry Number: EC 1.5.1.-

Grant and Affiliation Information for A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae.

AFFILIATION: Department of Chemistry and Biochemistry, University of Oklahoma, 620 Parrington Oval, Norman, Oklahoma 73019, USA.

Country: United States

AGENCY: United States NIGMS

GRANT: GM 071417

ACRONYM: GM

MEDLINETA: Biochemistry

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