IDENTIFICATION OF THE TRYPTOPHAN RESIDUE IN THE THIAMIN PYROPHOSPHATE BINDING-SITE OF MAMMALIAN PYRUVATE-DEHYDROGENASE
IDENTIFICATION OF THE TRYPTOPHAN RESIDUE IN THE THIAMIN PYROPHOSPHATE BINDING-SITE OF MAMMALIAN PYRUVATE-DEHYDROGENASE
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DOI：
10.1074/jbc.270.9.4570
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发表时间：
1995-03-03
影响因子：
4.8
通讯作者：
PATEL, MS
中科院分区：
文献类型：
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作者：
ALI, MS;SHENOY, BC;PATEL, MS
哺乳动物丙酮酸脱氢酶复合物的丙酮酸脱氢酶（E1）组分催化丙酮酸的氧化脱羧，形成乙酰基残基和还原当量，其依次转移到二氢硫辛酰乙酰转移酶和二氢硫辛酰胺脱氢酶组分。为了检查E1活性位点中的一个或多个乙酰基残基的作用，用Dahan特异性试剂N-溴代琥珀酰亚胺修饰该酶。修饰2个色氨酸残基/mol牛E1（四聚体α（2）β（2）中的12个）导致酶完全失活。通过与焦磷酸硫胺素（TPP）预孵育防止失活，表明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的强度，E1 + TPP在292 nm处的磁性圆二色性（MCD）光谱强度降低了约15%，因为该MCD带对色氨酸独特敏感且定量，最简单的解释是E1（α β二聚体）中存在的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时显著改变，伴随着非手性TPP大环在类似于263 nm处的光学活性的诱导。从CD研究还推断，在N-溴代琥珀酰亚胺处理后发生活性损失，而蛋白质的总体二级结构没有显著变化，通过在存在和不存在焦磷酸硫胺素的情况下用N-溴代琥珀酰亚胺修饰后的差异肽图谱分离出单一肽。发现从人E1产生的该肽对应于人E1 β的推导序列中的氨基酸残基116-143，表明人E1 β亚基中的色氨酸残基135在E1的活性位点中起作用。该色氨酸残基周围的氨基酸序列在几个物种的E1 β中是保守的，这表明该区域可能构成酶的结构和/或功能必需部分。
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 a 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 similar to 292 nm was decreased by similar to 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 similar to 263 nm for the nonchiral TPP macrocyle, 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 sequences 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.