IDENTIFICATION OF THE TRYPTOPHAN RESIDUE IN THE THIAMIN PYROPHOSPHATEBINDING-SITE OF MAMMALIAN PYRUVATE-DEHYDROGENASE

The pyruvate dehydrogenase (E1) component of the mammalian pyruvate de hydrogenase complex catalyzes the oxidative decarboxylation of pyruvat e with the formation of an acetyl residue and reducing equivalents, wh ich are transferred sequentially to the dihydrolipoyl acetyltransferas e and dihydrolipoamide dehydrogenase components, To examine the role o f tryptophanyl residue(s) in the active site of E1, the enzyme was mod ified with the tryptophan-specific reagent N-bromosuccinimide. Modific ation of 2 tryptophan residues/mol of bovine E1 (out of 12 in a tetram er alpha(2) beta(2)) resulted in complete inactivation of the enzyme. The inactivation was prevented by preincubation with thiamin pyrophosp hate (TPP), indicating that the modified tryptophan residue(s) is a pa rt 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 29 2 nm was decreased by similar to 15% for E1 + TPP relative to the inte nsity for E1 alone, Because this MCD band is uniquely sensitive to and quantitative for tryptophan, the simplest interpretation is that 1 ou t of 6 tryptophan residues present in E1 (alpha beta dimer) interacts with TPP. The natural circular dichroism (CD) spectrum of E1 is dramat ically altered upon binding TPP, with concomitant induction of optical activity at similar to 263 nm for the nonchiral TPP macrocyle, From C D Studies, it is also inferred that loss of activity following N-bromo succinimide treatment occurred without significant changes in the over all 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 pe ptide generated from human E1 was found to correspond to amino acid re sidues 116-143 in the deduced sequence of human E1 beta, suggesting th at the tryptophan residue 135 in the beta subunit of human E1 function s in the active site of E1. The amino acid sequences surrounding this tryptophan residue are conserved in E1 beta from several species, sugg esting that this region may constitute a structurally and/or functiona lly essential part of the enzyme.