Plaitakis A, Metaxari M, Shashidharan P
Department of Neurology, University of Crete, School of Health Sciences, Section of Medicine, Heraklion, Crete, Greece.
J Neurochem. 2000 Nov;75(5):1862-9. doi: 10.1046/j.1471-4159.2000.0751862.x.
Human glutamate dehydrogenase (GDH), an enzyme central to the metabolism of glutamate, is known to exist in housekeeping and nerve tissue-specific isoforms encoded by the GLUD1 and GLUD2 genes, respectively. As there is evidence that GDH function in vivo is regulated, and that regulatory mutations of human GDH are associated with metabolic abnormalities, we sought here to characterize further the functional properties of the two human isoenzymes. Each was obtained in recombinant form by expressing the corresponding cDNAs in Sf9 cells and studied with respect to its regulation by endogenous allosteric effectors, such as purine nucleotides and branched chain amino acids. Results showed that L-leucine, at 1.0 mM:, enhanced the activity of the nerve tissue-specific (GLUD2-derived) enzyme by approximately 1,600% and that of the GLUD1-derived GDH by approximately 75%. Concentrations of L-leucine similar to those present in human tissues ( approximately 0.1 mM:) had little effect on either isoenzyme. However, the presence of ADP (10-50 microM:) sensitized the two isoenzymes to L-leucine, permitting substantial enzyme activation at physiologically relevant concentrations of this amino acid. Nonactivated GLUD1 GDH was markedly inhibited by GTP (IC(50) = 0.20 microM:), whereas nonactivated GLUD2 GDH was totally insensitive to this compound (IC(50) > 5,000 microM:). In contrast, GLUD2 GDH activated by ADP and/or L-leucine was amenable to this inhibition, although at substantially higher GTP concentrations than the GLUD1 enzyme. ADP and L-leucine, acting synergistically, modified the cooperativity curves of the two isoenzymes. Kinetic studies revealed significant differences in the K:(m) values obtained for alpha-ketoglutarate and glutamate for the GLUD1- and the GLUD2-derived GDH, with the allosteric activators differentially altering these values. Hence, the activity of the two human GDH is regulated by distinct allosteric mechanisms, and these findings may have implications for the biologic functions of these isoenzymes.
人谷氨酸脱氢酶（GDH）是谷氨酸代谢的关键酶，已知分别存在由GLUD1和GLUD2基因编码的管家型和神经组织特异性同工型。鉴于有证据表明体内GDH功能受到调控，且人GDH的调控突变与代谢异常相关，我们在此进一步研究这两种人同工酶的功能特性。通过在Sf9细胞中表达相应的cDNA，以重组形式获得了每种同工酶，并研究了其受内源性变构效应物（如嘌呤核苷酸和支链氨基酸）的调控情况。结果显示，1.0 mM的L-亮氨酸可使神经组织特异性（源自GLUD2）酶的活性增强约1600%，使源自GLUD1的GDH活性增强约75%。与人组织中相似的L-亮氨酸浓度（约0.1 mM）对两种同工酶的影响都很小。然而，ADP（10 - 50 μM）的存在使两种同工酶对L-亮氨酸敏感，在该氨基酸的生理相关浓度下可实现显著的酶激活。未激活的GLUD1 GDH受到GTP的显著抑制（IC50 = 0.20 μM），而未激活的GLUD2 GDH对该化合物完全不敏感（IC50 > 5000 μM）。相反，由ADP和/或L-亮氨酸激活的GLUD2 GDH虽然对GTP抑制的敏感性低于GLUD1酶，但也会受到这种抑制，不过所需的GTP浓度要高得多。ADP和L-亮氨酸协同作用，改变了两种同工酶的协同曲线。动力学研究揭示，源自GLUD1和GLUD2的GDH在α-酮戊二酸和谷氨酸的Km值上存在显著差异，变构激活剂对这些值的影响也不同。因此，两种人GDH的活性受不同的变构机制调控，这些发现可能对这些同工酶的生物学功能具有重要意义。
