8 3-イソプロピルリンゴ酸脱水素酵素の特異的阻害剤と基質認識(口頭発表の部)

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3-Isopropylmalate dehydrogenase (IPMDH, EC 1.1.1.85) catalyzes an oxidation-decarboxylation reaction of (2R, 3S)-3-isopropylmalate (IPM) into 2-oxoisocaproate with the aid of NAD^+ in the penultimate step of the biosynthetic pathway of an essential amino acid L-leucine. We have been involved in the mechanistic and molecular recognition studies on IPMDH derived from the extremely thermophilic bacteria Thermus thermophlus HB8. The finding that 2-O-methyl-3-isopropylmalate (1) is an uncompetitive inhibitor of IPMDH, prompted to design conformationally restricted substrate analogs, in which the hydroxy oxygen is intramolecularly bound to an isopropyl carbon to form a ring structure. The oxirane 2 was the most inhibitory among those synthesized. IPMDH appeared to recognize preferentially the anticonformation of the butanedioic acid structure. (2R, 3S)-3-vinylmalic acid (VM) was designed, as a mechanism-based inhibitor. The synthesis of VM was pursued in 6 steps from diethyl (R)-malate. Besides its weak activity as a substrate, VM appeared to be a competitive and mechanism-based inhibitor (K_i values 25 nM) for IPMDH as deduced from the time-dependent and kinetic analyses. By GC-MS and ^1H-NMR analysis of the enzyme reaction of VM, 2-oxo-3-pentenoate (7) was detected as the product. Furthermore, we synthesized deuterium-labeled VM (VM-d_3) and monitored the enzyme reaction by ^2H-NMR spectroscopy. While the signals of VM-d_3 (at δ_D 5.1 and 5.8 ppm) disappeared by consumption of VM-d_3, the signals which derived from 7 emerged at δ_D 2.0 and 7.1 ppm. These results suggest that the transient covalent bond, which was formed between IPMDH and 7, inhibits the IPMDH reaction. Arg94Lys mutant of IPMDH was prepared by site-directed mutagenesis. The mutant enzyme had the same Km value as the native enzyme. But its catalytic efficiency lowered 10^<-2> times as compared with wild-type IPMDH. Arg94 of IPMDH appeared to stabilize the transition state of the IPMDH reaction and participate in the catalytic efficiency.
天然有機化合物討論会の論文
1998-08-31

8 3-イソプロピルリンゴ酸脱水素酵素の特異的阻害剤と基質認識(口頭発表の部)

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