Ali M S, Shenoy B C, Eswaran D, Andersson L A, Roche T E, Patel M S
Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
J Biol Chem. 1995 Mar 3;270(9):4570-4. doi: 10.1074/jbc.270.9.4570.
The pyruvate dehydrogenase (E1) component of the mammalian pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate with the formation of an acetyl residue and reducing equivalents, which are transferred sequentially to the dihydrolipoyl acetyltransferase and dihydrolipoamide dehydrogenase components. To examine the role of tryptophanyl residue(s) in the active site of E1, the enzyme was modified with the tryptophan-specific reagent N-bromosuccinimide. Modification of 2 tryptophan residues/mol of bovine E1 (out of 12 in a tetramer alpha 2 beta 2) resulted in complete inactivation of the enzyme. The inactivation was prevented by preincubation with thiamin pyrophosphate (TPP), indicating that the modified tryptophan residue(s) is part of the active site of this enzyme. Fluorescence studies showed that thiamin pyrophosphate interacts with tryptophan residue(s) of E1. The magnetic circular dichroism (MCD) spectral intensity at approximately 292 nm was decreased by approximately 15% for E1 + TPP relative to the intensity for E1 alone. Because this MCD band is uniquely sensitive to and quantitative for tryptophan, the simplest interpretation is that 1 out of 6 tryptophan residues present in E1 (alpha beta dimer) interacts with TPP. The natural circular dichroism (CD) spectrum of E1 is dramatically altered upon binding TPP, with concomitant induction of optical activity at approximately 263 nm for the nonchiral TPP macrocycle. From CD studies, it is also inferred that loss of activity following N-bromosuccinimide treatment occurred without significant changes in the overall secondary structure of the protein. A single peptide was isolated by differential peptide mapping in the presence and absence of thiamin pyrophosphate following modification with N-bromosuccinimide. This peptide generated from human E1 was found to correspond to amino acid residues 116-143 in the deduced sequence of human E1 beta, suggesting that the tryptophan residue 135 in the beta subunit of human E1 functions in the active site of E1. The amino acid sequence surrounding this tryptophan residue are conserved in E1 beta from several species, suggesting that this region may constitute a structurally and/or functionally essential part of the enzyme.
哺乳动物丙酮酸脱氢酶复合体中的丙酮酸脱氢酶（E1）组分催化丙酮酸的氧化脱羧反应，生成乙酰基残基和还原当量，这些物质会依次转移至二氢硫辛酰乙酰基转移酶和二氢硫辛酰胺脱氢酶组分。为了研究色氨酸残基在E1活性位点中的作用，用色氨酸特异性试剂N - 溴代琥珀酰亚胺对该酶进行修饰。每摩尔牛E1（四聚体α2β2中的12个色氨酸残基）中有2个色氨酸残基被修饰，导致该酶完全失活。用焦磷酸硫胺素（TPP）预孵育可防止这种失活，这表明被修饰的色氨酸残基是该酶活性位点的一部分。荧光研究表明，焦磷酸硫胺素与E1的色氨酸残基相互作用。相对于单独的E1，E1 + TPP在约292nm处的磁圆二色性（MCD）光谱强度降低了约15%。由于该MCD谱带对色氨酸具有独特的敏感性且是定量的，最简单的解释是E1（αβ二聚体）中6个色氨酸残基中的1个与TPP相互作用。E1的天然圆二色性（CD）光谱在结合TPP后会发生显著变化，对于非手性的TPP大环，在约263nm处会伴随光学活性的诱导。从CD研究还可推断，N - 溴代琥珀酰亚胺处理后活性丧失，但蛋白质的整体二级结构没有明显变化。在用N - 溴代琥珀酰亚胺修饰后，通过在有和没有焦磷酸硫胺素存在的情况下进行差异肽图谱分析，分离出了一个单一肽段。发现从人E1产生的这个肽段对应于人E1β推导序列中的氨基酸残基116 - 143，这表明人E1β亚基中的色氨酸残基135在E1的活性位点中起作用。这个色氨酸残基周围的氨基酸序列在几种物种的E1β中是保守的，这表明该区域可能构成了该酶结构和/或功能上必不可少的部分。
