Characterization of the carboxylate delivery module of transcarboxylase: following spontaneous decarboxylation of the 1.3S-CO2- subunit by NMR and FTIR spectroscopies.
Transcarboxylase (TC) is a multisubunit enzyme that catalyzes the transfer of a carboxylate group from methylmalonyl-CoA (MMCoA) to pyruvate. The CO2- group is shuttled between the MMCoA and pyruvate binding sites by a biotin cofactor, covalently linked to the 1.3S subunit. Fully carboxylated 1.3S can be prepared in vitro using 1.3S, MMCoA, and catalytic amounts of the TC's MMCoA-binding subunit. The 1.3S-CO2- intermediate decarboxylates spontaneously over a period of hours, and this process was characterized by 1D and 2D NMR and FTIR spectroscopies. The NMR data yielded a first-order kinetic constant of 1.4 x 10(-3) min(-1) for the spontaneous decarboxylation. This rate was calculated from the 1D NMR spectrum by measuring the reappearance of biotin's ureido NH protons and the disappearance of peaks at 6.99 and 7.67 ppm assigned to Asn-8 and/or Asn-24 from the 1.3S's N-terminus. The latter peaks are absent in the 1D spectrum of non-carboxylated 1.3S due to exchange between two or more conformations within the N-terminus causing line broadening. It is proposed that interactions between the biotin-CO2- and the N-terminal amino acids perturb this conformational equilibrium causing some N-terminal residues to appear in the 1D NMR spectrum of the carboxylated form. Further details are apparent from a comparison of the 2D spectra of the 1.3S-CO2- and 1.3S proteins, where carboxylation causes several peaks from the C-terminal half to shift as well as the appearance of resonances due to some residues located at the N-terminal half of the protein. FTIR difference spectra were used also to follow spontaneous decarboxylation of the 1.3S-CO2-. For the carboxylated 1.3S, the difference spectra provided the vibrational signature of the CO2- on the biotin ring. A doublet was identified at 1695 and 1699 cm(-1) that increased in intensity with increasing t. This is assigned to an antisymmetric stretching vibration of the CO2- group bound to biotin on the 1.3S protein. Its position and profile provide further evidence for interactions occurring between the biotin-CO2- group and the 1.3S protein. These studies demonstrate the highly mobile, "poised" nature of the 1.3S protein engineered for its role as a CO2- translocator.
Rivera-Hainaj
Rosa E
RE
Department of Biochemistry and Department of Physiology and Biophysics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935, USA.
Pusztai-Carey
Marianne
M
Venkat Reddy
D
D
Choowongkomon
Kiattawee
K
Sönnichsen
Frank D
FD
Carey
Paul R
PR
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry / Q000378:metabolism; D001710:Biotin / Q000737:chemistry / Q000378:metabolism; D002245:Carbon Dioxide / Q000737:chemistry*; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry* / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry* / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D003653:Decarboxylation; D054889:Fatty Acid Synthase, Type II; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011424:Propionibacterium / Q000201:enzymology; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods
D002264:Carboxylic Acids; D002352:Carrier Proteins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D002245:Carbon Dioxide; D001710:Biotin; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein