CRYSTALLOGRAPHIC ANALYSIS OF SUBSTRATE BINDING AND CATALYSIS IN DIHYDROLIPOYL TRANSACETYLASE (E2P)

The catalytic domain of dihydrolipoyl transacetylase (E2pCD) forms the core of the pyruvate dehydrogenase multienzyme complex and catalyzes the acetyltransferase reaction using acetylCoA as acetyl donor and dihydrolipoamide (Lip(SH)2) as acceptor. The crystal structures of six complexes and derivatives of Azotobacter vinelandii E2pCD were solved. The binary complexes of the enzyme with CoA and Lip-(SH)2 were determined at 2.6- and 3.0-angstrom resolutions, respectively. The two substrates are found in an extended conformation at the two opposite entrances of the 30 angstrom long channel which runs at the interface between two 3-fold-related subunits and forms the catalytic center. The reactive thiol groups of both substrates are within hydrogen-bond distance from the side chain of His 610. This fact supports the indication, derived from the similarity with chloramphenicol acetyl transferase, that the histidine side chain acts as general-base catalyst in the deprotonation of the reactive thiol of CoA. The conformation of Asn 614 appears to be dependent on the protonation state of the active site histidine, whose function as base catalyst is modulated in this way. Studies on E2pCD soaked in a high concentration of dithionite lead to the structure of the binary complex between E2pCD and hydrogen sulfite solved at 2.3-angstrom resolution. It appears that the anion is bound in the middle of the catalytic center and is therefore capable of hosting and stabilizing a negative charge, which is of special interest since the reaction catalyzed by E2pCD is thought to proceed via a negatively charged tetrahedral intermediate. The structure of the binary complex between E2pCD and hydrogen sulfite suggests that transition-state stabilization can be provided by a direct hydrogen bond between the side chain of Ser 558 and the oxy anion of the putative intermediate. In the binary complex with CoA, the hydroxyl group of Ser 558 is hydrogen bonded to the nitrogen atom of one of the two peptide-like units of the substrate. Thus, CoA itself is involved in keeping the Ser hydroxyl group in the proper position for transition-state stabilization. Quite unexpectedly, the structure at 2.6-angstrom resolution of a ternary complex in which CoA and Lip(SH)2 are simultaneously bound to E2pCD reveals that CoA has an alternative, nonproductive binding mode. In this abortive ternary complex, CoA adopts a helical conformation with two intramolecular hydrogen bonds and the reactive sulfur of the pantetheine arm positioned 12 angstrom away from the active site residues involved in the transferase reaction. The same CoA conformation is observed in the ternary complex with CoA and dithiothreitol. The latter behaves like a substrate analogue which binds in the Lip(SH)2 binding pocket. The strict conservation of the active site residues among the transacylases forming the core of the branched-chain oxo acid dehydrogenase and the oxoglutarate dehydrogenase multienzyme complexes indicates that these enzymes all share the same catalytic mechanism as dihydrolipoyl transacetylase. The investigation of the three-dimensional structure of E2pCD combined with the analysis of the amino acid sequences suggests that the nature and size of only a few side chains might be the major determinants for tuning substrate specificity among the various transacylases.