Supporting role of lysine 13 and glutamate 16 in the acid-base mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

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• 作者：Vidya Prasanna Kumar ; Ann H. West ; Paul F. Cook ; ^pcook@ou.edu
• 关键词：Lysine biosynthesis ; Saccharopine dehydrogenase ; Site-directed mutagenesis ; Isotope effects ; pH-rate profiles ; Enzyme mechanism
• 刊名：Archives of Biochemistry and Biophysics
• 出版年：2012
• 期刊代码：116_00039861
• 类别：ch
• 出版时间：1 June, 2012
• 卷：522
• 期：1
• 页码：57-61
• 文件大小：498 K

摘要

Saccharopine dehydrogenase (SDH) catalyzes the NAD⁺ dependent oxidative deamination of saccharopine to form lysine (Lys) and 伪-ketoglutarate (伪-kg). The active site of SDH has a number of conserved residues that are believed important to the overall reaction. Lysine 13, positioned near the active site base (K77), forms a hydrogen bond to E78 neutralizing it, and contributing to setting the pK_a of the catalytic residues to near neutral pH. Glutamate 16 is within hydrogen bond distance to the N蔚 atom of R18, which has strong H-bonding interactions with the 伪-carboxylate and 伪-oxo groups of 伪-kg. Mutation of K13 to M and E16 to Q decreased k_cat by about 15-fold, and primary and solvent deuterium kinetic isotope effects measured with the mutant enzymes indicate hydride transfer is rate limiting for the overall reaction. The pH-rate profiles for K13M exhibited no pH dependence, consistent with an increase in negative charge in the active site resulting in the perturbation in the pK_as of catalytic groups. Elimination of E16 affects optimal positioning of R18, which is involved in binding and holding 伪-kg in the correct conformation for optimum catalysis. In agreement, a 螖螖G掳鈥?of 2.60 kcal/mol is estimated from the change in K_伪-kg for replacing E16 with Q.