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

Saccharopine dehydrogenase (N6-(glutaryl-2)-L-lysine: NAD oxidoreductase (L-lysine forming); (EC 1.5.1.7)) (SDH1) catalyzes the final step in the α-aminoadipate pathway (AAA) for the de novo synthesis of L-lysine in fungi (1, 2). The enzyme catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate α-Kg and lysine using NAD as an oxidant, eq. 1 (1). (1) The histidine-tagged SDH from S. cerevisiae has been overexpressed in Escherichia coli and purified to about 98% using a Ni-NTA resin (3). The kinetic mechanism of the SDH reaction has recently been determined from initial velocity studies in the absence and presence of product and dead-end inhibitors (3). The SDH catalyzes an ordered mechanism in which NAD is the first substrate bound followed by saccharopine in the physiologic reaction direction. In the reverse reaction direction, NADH adds to enzyme first, and there is no preference for binding of α-Kg and lysine at pH 7.0. The presence of the AAA pathway for lysine biosynthesis has been demonstrated in several yeasts and molds including human pathogens such as Candida albicans, Cryptococcus neoformas, and Aspergillus fumigatus and plant pathogens such as Magnaporthe grisea (4, 5). The uniqueness of the AAA pathway makes it a target for the rapid detection, growth control of pathogenic yeasts and molds, and for antifungal drug design. Toward this end, it is important to determine the chemical mechanism of the enzyme. A first step in this process is the elucidation of the acid-base mechanism of SDH. Preliminary information on the mechanism of the SDH has been obtained by other groups, and this will be summarized briefly. The S. cerevisiae enzyme has pH optima of 10.0 in the direction of lysine formation, and 7.0 in the direction of saccharopine formation (2). The pH dependence of V/KLys is bell shaped giving pKa values of 6.3 and 8.0, while the V/Kα-Kg profile exhibits a single pKa of 8.4 on the basic side (6). The V/KSacc pH-rate profile exhibits two pKa values of 6.0 and 7.1 on the acid side (6). A series of chemical modification experiments have also been carried out suggesting the presence of essential cysteine (7), histidine (8), lysine (9), and arginine residues (10) that may be involved in substrate-binding and/or catalysis. On the basis of the above, a chemical mechanism has been proposed for SDH in which saccharopine is first oxidized to e-N-glutariminyl-L-lysine. A general base then activates water for attack to form a carbinolamine intermediate, which collapses to give the products, L-lysine and α-Kg via the participation of a general acid and a general base (6). Previous studies of the chemical mechanism of SDH are incomplete and the identity of the groups involved is based on the observed pKa values, their temperature dependence, and chemical modification studies. In this paper, the pH dependence of the kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects are used to probe the chemical mechanism of SDH. A proton shuttle mechanism is proposed and data are discussed in terms of potential rate limiting steps along the reaction pathway.