Phosphonate analogs of pyruvate and 2-oxoglutarate are established specific inhibitors of cognate 2-oxo acid dehydrogenases. The present work develops application of this class of compounds to specific in vivo inhibition of 2-oxoglutarate dehydrogenase (OGDH) and its isoenzyme, 2-oxoadipate dehydrogenase (OADH). The isoenzymes-enriched preparations from the rat tissues with different expression of OADH and OGDH are used to characterize their interaction with 2-oxoglutarate (OG), 2-oxoadipate (OA) and the phosphonate analogs. Despite a 100-fold difference in the isoenzymes ratio in the heart and liver, similar Michaelis saturations by OG are inherent in the enzyme preparations from these tissues (KmOG = 0.45 ± 0.06 and 0.27 ± 0.026 mM, respectively), indicating no significant contribution of OADH to the OGDH reaction, or similar affinities of the isoenzymes to OG. However, the preparations differ in the catalysis of OADH reaction. The heart preparation, where OADH/OGDH ratio is ≈ 0.01, possesses low-affinity sites to OA (KmOA = 0.55 ± 0.07 mM). The liver preparation, where OADH/OGDH ratio is ≈ 1.6, demonstrates a biphasic saturation with OA: the low-affinity sites (Km,2OA = 0.45 ± 0.12 mM) are similar to those of the heart preparation; the high-affinity sites (Km,1OA = 0.008 ± 0.001 mM), revealed in the liver preparation only, are attributed to OADH. Phosphonate analogs of C5-C7 dicarboxylic 2-oxo acids inhibit OGDH and OADH competitively to 2-oxo substrates in all sites. The high-affinity sites for OA are affected the least by the C5 analog (succinyl phosphonate) and the most by the C7 one (adipoyl phosphonate). The opposite reactivity is inherent in both the low-affinity OA-binding sites and OG-binding sites. The C6 analog (glutaryl phosphonate) does not exhibit a significant preference to either OADH or OGDH. Structural analysis of the phosphonates binding to OADH and OGDH reveals the substitution of a tyrosine residue in OGDH for a serine residue in OADH among structural determinants of the preferential binding of the bulkier ligands to OADH. The consistent kinetic and structural results expose adipoyl phosphonate as a valuable pharmacological tool for specific in vivo inhibition of the DHTKD1-encoded OADH, a new member of mammalian family of 2-oxo acid dehydrogenases, up-regulated in some cancers and associated with diabetes and obesity.

丙酮酸和2-氧代戊二酸的膦酸酯类似物是相关的2-氧代酸脱氢酶的特异性抑制剂。本工作开发了这类化合物在特定领域的应用体内抑制2-氧代戊二酸脱氢酶（OGDH）及其同工酶2-氧代己二酸脱氢酶（OADH）。用大鼠组织中富含OADH和OGDH的不同表达的富含同工酶的制剂来表征它们与2-氧代戊二酸酯（OG），2-氧代己二酸酯（OA）和膦酸酯类似物的相互作用。尽管心脏和肝脏中同工酶的比率相差100倍，但来自这些组织的酶制剂中固有的OG相似的米氏臭味是固有的（ķ米ØG=分别为0.45±0.06和0.27±0.026 mM），这表明OADH对OGDH反应无明显贡献，或同工酶对OG的相似亲和力。然而，制备方法在OADH反应的催化上有所不同。OADH / OGDH比约为0.01的心脏制剂对OA具有低亲和力位点（ķ米Ø一种= 0.55±0.07 mM）。OADH / OGDH比约为1.6的肝脏制剂显示OA具有两相饱和性：低亲和力位点（ķ米，2Ø一种= 0.45±0.12 mM）类似于心脏制剂; 高亲和力网站（ķ米，1个Ø一种= 0.008±0.001 mM）（仅在肝脏制剂中显示）归因于OADH。C5-C7二羧酸2-氧代酸的膦酸酯类似物在所有位点均对2-氧代底物具有竞争性抑制OGDH和OADH。OA的高亲和力位点受C5类似物（琥珀酰膦酸酯）影响最小，而受C7类似物（己二酰基膦酸酯）影响最大。在低亲和力的OA结合位点和OG结合位点中固有的是相反的反应性。C6类似物（戊二酸酯膦酸酯）对OADH或OGDH均没有明显的偏爱。对与OADH和OGDH结合的膦酸酯的结构分析表明，在较大的配体与OADH优先结合的结构决定因素中，OGDH中的酪氨酸残基取代了OADH中的丝氨酸残基。体内 抑制 DHTKD1-编码的OADH，它是哺乳动物2-氧酸脱氢酶家族的新成员，在某些癌症中表达上调，并与糖尿病和肥胖症有关。