KINETICS AND SPECIFICITY OF REDUCTIVE ACYLATION OF MILD-TYPE AND MUTATED LIPOYL DOMAINS OF 2-OXO-ACID DEHYDROGENASE COMPLEXES FROM AZOTOBACTER-VINELANDII

The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2-oxo-acid dehydrogenase complexes , catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild-type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 12 6 s(-1), corresponds well with the turnover rate derived from steady-s tate measurements. Deacetylation was rapid and specific for coenzyme A . No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex-boun d lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This g ain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex , and illustrates the efficiency of substrate channeling in the multie nzyme complex. The 2-oxo-acid dehydrogenases exhibit specificity for l ipoyl domains in the reductive acylation reaction. The A. vinelandii l ipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2-oxogluta rate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl dom ain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinyla ted by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2-oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants o f the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl do main sequences and structures, an exposed loop of the A. vinelandii 2- oxoglutarate dehydrogenase complex lipoyl domain was subjected to muta genesis. Although the reductive acylation experiments of mutants of th e lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.