Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) - evidence for tertiary and quaternary interactions.
The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the beta domain at >20 A from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the alpha domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together 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 E1 component 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 cofactors.
Jordan
F
F
Department of Chemistry, Rutgers, the State University, Newark, NJ 07102, USA. frjordan@andromeda.rutgers.edu
Nemeria
N
N
Guo
F
F
Baburina
I
I
Gao
Y
Y
Kahyaoglu
A
A
Li
H
H
Wang
J
J
Yi
J
J
Guest
J R
JR
Furey
W
W
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D008274:Magnesium / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011768:Pyruvate Dehydrogenase Complex / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000378:metabolism*
D011768:Pyruvate Dehydrogenase Complex; D019289:Pyruvic Acid; D011767:Pyruvate Decarboxylase; D008274:Magnesium; D013835:Thiamine Pyrophosphate