Identification of the Tryptophan Residue in the Thiamin Pyrophosphate Binding Site of Mammalian Pyruvate Dehydrogenase (*)

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 α2β2) 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 ∼292 nm was decreased by ∼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 (αβ 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 ∼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 E1β, suggesting that the tryptophan residue 135 in the β subunit of human E1 functions in the active site of E1. The amino acid sequences surrounding this tryptophan residue are conserved in E1β from several species, suggesting that this region may constitute a structurally and/or functionally essential part of the enzyme.