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Saccharopine, an Intermediate of the Aminoadipic Acid Pathway of Lysine Biosynthesis

III. AMINOADIPIC SEMIALDEHYDE-GLUTAMATE REDUCTASE

From the ¹ From the Laboratory of Biochemistry, Department of Dairy Science, University of Illinois, Urbana, Illinois 61803

An enzyme, aminoadipic semialdehyde-glutamate reductase (-N-(L-glutaryl-2)-L-lysine: NAD⁺(P) oxidoreductase (L-2-aminoadipate-semialdehyde forming)), of concern in lysine synthesis from -aminoadipic acid in yeast was purified over 100-fold from bakers' yeast by procedures which included ammonium sulfate precipitation, chromatography on calcium phosphate gel cellulose and diethylaminoethyl cellulose, and negative adsorption on alumina gel C. The enzyme was shown to catalyze the following reversible reaction.

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At a neutral pH, saccharopine was formed only when aminoadipic semialdehyde, glutamate, and reduced nicotinamide adenine dinucleotide were all present. NADH was much less active than NADPH as the coenzyme. In this system, when aminoadipic semialdehyde (3) or glutamate were radioactive, saccharopine, recognized by its behavior on paper electrophoresis, was radioactive. At an alkaline pH, e.g. pH 9.75, saccharopine was cleaved oxidatively either by NADP⁺ or NAD⁺ to form aminoadipic semialdehyde, as characterized by its o-aminobenzaldehyde adduct, and glutamate, detected with glutamic dehydrogenase. In this latter instance, the amount of glutamate formed corresponded to the amount of NADP⁺ reduced. Aminoadipic semialdehyde-glutamate reductase is a sulfhydryl enzyme having an s_20,w value of 4.9 and a molecular weight of approximately 73,000 as estimated by sucrose density gradient centrifugation. The Michaelis constants for saccharopine and NADP⁺ are 0.92 mM and 0.022 mM, respectively.

Aminoadipic semialdehyde-glutamate reductase, when coupled with saccharopine dehydrogenase which will be discussed in the accompanying paper (9), functions to synthesize lysine from aminoadipic semialdehyde via the stable intermediate, saccharopine.

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