Four enzymes of the lysine biosynthetic pathway of Candida maltosa were investigated and the regulatory pattern was established. a-Aminodipate reductase ( M , 160,000), saccharopine reductase ( M , 90,000) and saccharopine dehydrogenase (Mi-45,000) were separated from each other.
Homocitrate synthase was sensitive to the separation methods used. The formation of saccharopine rcductase was constitutive, whereas the synthesis of homocitrate synthase, a-aminoadipate reductase and saccharopine dehydrogenase were regulated by the general control of amino acid biosynthesis. L-Lysine (Ki = 5.0 mM), L-thialysine (Ki = 15 mM) and oL-a-aminoadipate (Ki = 5.1 miv) inhibited homocitrate synthase, the first enzyme of the pathway. The inhibition was competitive with respect to 2-oxoglutarate. The Km-values of the enzyme were estimated to be 0.033 miw for acetyl-CoA and 0.025 mM for 2-oxoglutarate. The MICHAELIS constants of the a-aminoadipate reductase were calculated to be 0.20 mM for a-nminoadipate, 0.50 mM for ATP, 0.10 mM for NADPH and 3.8 mM for MgCI,. The Km-values of the saccharopine reductase were 0.55 mM for saccharopine and 0.13 mM for NADP+, respectively. The values of the saccharopine dehydrogenase were estimated to be 1.6 mM for L-lysine, 0.66 mM for 2-oxoglutarate and 0.23 mni for NADH. Fnrthcrmore, a number of amino acids were found to be effectors of the enzyme reactions.
In nature lysine is biosynthesized via two mutally exclusive pathways. Bacteria, soiiie Yhycoinycetes and green plants utilize the dianiinopimelate pathway for its synt,hesis. In contrast, the a-aminoadipate pathway is used by Euglenids, some Phycomycetes, yeasts and higher fungi.
The first intermediate of the a-aminoadipate pathway, homocitrate, is formed by condensation of acetyl-CoA with 2-oxoglutarate. This condensation is mediated by homocitrate synthase which is the most important site of control in the lysine patlzway of Saccharomyces cerevisiae ( TUCCI and CECI 1972), Yarrowia lipolytica ( GAILLAB-DIN et al. 1976), Candida pelliculosa (TAKENOUCHI et al. 1981) and Neurospora crassrc (HOGG and BROQUIST 1968). The eight biosynthetic steps following this reaction are not controlled by feedback inhibition.
This paper described some physical characteristics and regulatory patterns of folir of the enzymes involved in lysine biosynthetic pathway (homocitrate synthase, a- aminoadipate reductase, saccharopine reductase and dehydrogenase) of the industrially important n-alkane-utilizing yeast Candida maltosa. Results indicate that in v i ~o only homocitrate synthase is ieedback-inhibited by lysine and that the synthesis of three enzymes responds to general amino acid regulation.