Involvement of tryptophans at the catalytic and subunit-binding domains of transcarboxylase.
Transcarboxylase from Propionibacterium shermanii is a multisubunit enzyme. It consists of one central hexameric subunit to which six outer dimeric subunits are attached through twelve biotinyl subunits. Both the central and the outer subunits are multi-tryptophan (Trp) proteins, and each contains 5 Trps per monomer. The roles of the Trps during catalysis and assembly of the enzyme have been studied by using N-bromosuccinimide (NBS) oxidation as a probe. Modification of approximately 10 Trps of the total 90 Trps of the intact enzyme results in loss of activity. Both the substrates, viz., methylmalonyl-CoA and pyruvate, afford protection (approximately 50%) against inactivation caused by NBS. Analyses of tryptic peptide maps and intrinsic fluorescence studies have indicated that modification of 10 Trps of the whole enzyme does not cause extensive conformational changes. Therefore, the Trps appear to be essential for catalytic activity. NBS modification of the individual subunits at pH 6.5 has demonstrated differential reactivity of their Trps. Modification of the exposed/reactive Trps of either one of the subunits significantly affects the subunit assembly with the complementary unmodified subunits to form active enzyme. It is proposed that Trps are involved at the subunit-binding domains of either the central or the outer subunit of transcarboxylase, in addition to those critical for catalysis.
Kumar
G K
GK
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Beegen
H
H
Wood
H G
HG
D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D019878:Carboxyl and Carbamoyl Transferases; D010084:Oxidation-Reduction; D011424:Propionibacterium / Q000201:enzymology; D011487:Protein Conformation; D014166:Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014364:Tryptophan
D014364:Tryptophan; D014166:Transferases; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D001974:Bromosuccinimide