REGULATION OF THIAMIN DIPHOSPHATE-DEPENDENT 2-OXO ACID DECARBOXYLASESBY SUBSTRATE AND THIAMIN DIPHOSPHATE.MG(II) - EVIDENCE FOR TERTIARY AND QUATERNARY INTERACTIONS

The regulatory mechanism of substrate activation in yeast pyruvate dec arboxylase is triggered by the interaction of pyruvic acid with C221 l ocated on the beta domain at >20 Angstrom from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitut ions were made at H92 on the a domain, across the domain divide from C 221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substit utions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, inclu ding the Hill coefficient, provide useful ir.sight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 togethe r appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg (II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the Il compon ent of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg (II) ligands are very important to activation of the enzymes by cofact ors. (C) 1998 Elsevier Science B.V. All rights reserved.