Protein Engineering Studies on Structure and Function of Amino Acid Dehydrogenase

本研究取得了以下结果。（a）通过磷酸吡哆醛的化学修饰鉴定亮氨酸脱氢酶中的活性位点赖氨酸通过与5 ′-磷酸吡哆醛（PLP）温育，然后用硼氢化钠还原，使来自嗜热脂肪芽孢杆菌的亮氨酸脱氢酶失活。当L-亮氨酸和NAD^+共存时，失活被完全抑制。几个PLP分子确实被纳入一摩尔的酶亚基伴随着失活。从标记蛋白质的蛋白水解消化物中分离的荧光肽的序列分析显示，Lys 80、Lys 91、Lys 206和Lys 265被标记。Lys 8 O最主要被标记，并且在L-亮氨酸和NAD^+存在下，被特异性地保护而不被标记。此外，在残留活性和掺入Lys 8 O的PLP量之间观察到约1：1的线性关系。Lys 8 O是保守的（B ...更多信息 亮氨酸脱氢酶活性位点Lys 80的定点突变功能研究亮氨酸脱氢酶活性位点Lys 80通过定点突变被Ala、Arg或Gln取代。纯化的Lys 8 O突变体酶在氧化脱氨中显示出明显低的V值（野生型酶的0.2-1.6%），但<max>在还原胺化中显示出可变的V值（Lys 8 O-Ala，89% ; Lys 8 O-Gln，23% ;和Lys 8 O-Arg，0.4%）。Lys 8 O-Ala和Lys 8 O-Gln突变体酶对α-酮-异己酸的Km值显著增加，而所有突变体酶对NAD^+和NADH的Km值与野生型酶相似。异己酸酯是α-酮-异己酸的非底物类似物，竞争性抑制野生型和Lys 8 O-Ala突变体酶的反应，其K_i值远高于野生型酶的α-酮-异己酸的K_m值，这表明Lys 8 O的 *-氨基和α-酮基-异-己酸酯在酮酸底物的结合中起重要作用。外源添加的伯胺（包括铵离子）对Lys 8 OAla突变酶催化的反应有明显的促进作用，表明添加的伯胺可以部分取代Lys 8 O上的 *-氨基的功能，这取决于它们的碱性（pK_a值）和分子体积。这些结果表明，Lys 8 O活性中心的 *-氨基在亮氨酸脱氢酶的催化过程中起着一般酸碱基团的作用。少

The following results have been obtained by the present studies.(a)Identification of an Active-Site Lysine in Leucine Dehydrogenase by Chemical Modification with Pyridoxal PhosphateLeucine dehydrogenase from Bacillus stearothermophilus was inactivated by incubation with pyridoxal 5'-phosphate(PLP)followed by reduction with sodium borohydride. The inactivation was completely retarded in the copresence of L-leucine and NAD^+. Several PLP molecules were indeed incorporated into one mol of the enzyme subunit concomitantly with the inactivation. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the labeled protein revealed that Lys80, Lys91, Lys206, and Lys265 were labeled. Lys8O was most predominantly labeled and, in the presence of L-leucine and NAD^+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1 : 1 was observed between the residual activity and the amount of PLP incorporated into Lys8O. Lys8O is conserved(b … More )Functional Analysis of Active-Site Lysine 80 in Leucine Dehydrogenase by Site-Directed MutagenesisLys80 of leucine dehydrogenase has been replaced by Ala, Arg, or Gln by sitedirected mutagenesis. The Lys8O mutant enzymes purified to homogeneity showed markedly low V values(0.2-1.6% of that of the wild-type enzyme)in the oxidative deamination, but variable_<max> values in the reductive amination(Lys8O-Ala, 89% ; Lys8O-Gln, 23% ; and Lys8O-Arg, 0.4% of that of the wild-type enzyme). The Lys8O-Ala and Lys8O-Gln mutant enzymes exhibited considerably increased K_m values for alpha-keto-iso-caproate, whereas the K_m values for NAD^+ and NADH of all the mutant enzymes were similar to those of the wild-type enzyme. iso-Caproate, a non-substrate analogue of alpha-keto-iso-caproate, competitively inhibited the reaction of both the wild-type and Lys8O-Ala mutant enzymes with K_i values much higher than the K_m value for alpha-keto-iso-caproate of the wild-type enzyme, suggesting the importance of an electrostatic interaction between the *-amino group of Lys8O and the alpha-carbonyl group of alpha-keto-iso-caproate in the binding of the keto acid substrate. Exogenously added primary amines including ammonium ion remarkably accelerated the reaction catalyzed by the Lys8OAla mutant enzyme, indicating that the added amines can partially replace the function of *-amino group of Lys8O depending on both their basicities (pK_a values) and molecular volumes. These results lead to the conclusion that the *-amino group of the active site Lys8O ftinctions as a general acid-base group in the catalysis of leucine dehyarogenase. Less