Substrate binding induces a cooperative conformational change in the 12S subunit of transcarboxylase: Raman crystallographic evidence.
The 12S subunit of transcarboxylase is a 338 000 Da hexamer that transfers carboxlylate from methylmalonyl-CoA (MM-CoA) to biotin; in turn, the biotin transfers the carboxylate to pyruvate on another subunit, the 5S. Here, Raman difference microscopy is used to study the binding of substrate and product, and their analogues, to single crystals of 12S. A single crystal is the medium of choice because it provides Raman data of unprecedented quality. Crystalline ligand-protein complexes were formed by cocrystallization or by the soaking in/soaking out method. Raman difference spectra were obtained by subtracting the spectrum of the apo crystal from that of a crystal with the substrate or product bound. Raman difference spectra from crystals with the substrate bound are dominated by bands from the protein's amide bonds and aromatic side chain residues. In contrast, Raman difference spectra involving the product, propionyl-CoA, are dominated by modes from the ligand. These results show that substrate binding triggers a conformational change in 12S, whereas product binding does not. The conformational change involves an increase in the amount of alpha-helix since markers for this secondary structure are prominent in the difference spectra of the substrate complex. The number of MM-CoA ligands bound per 12S hexamer can be gauged from the intensity of the MM-CoA Raman features and the fact that the protein concentration in the crystals is known from X-ray crystallographic data. Most crystal samples had six MM-CoAs per hexamer although a few, from different soaking experiments, contained only 1-2. However, both sets of crystals showed the same degree of protein conformational change, indicating that the change induced by the substrate is cooperative. This effect allowed us to record the Raman spectrum of bound MM-CoA without interference from protein modes; the Raman spectrum of a 12S crystal containing 2 MM-CoA ligands per hexamer was subtracted from the Raman spectrum of a 12S crystal containing six MM-CoA ligands per hexamer. The conformational change is reversible and can be controlled by soaking out or soaking in the ligand, using either concentrated ammonium sulfate solutions or the solution used in the crystallization trials. Malonyl-CoA also binds to 12S crystals and brings about conformational changes identical to those seen for MM-CoA; in addition, butyryl-CoA binds and behaves in a manner similar to propionyl-CoA. These data implicate the -COO- group on MM-CoA (that is transferred to biotin in the reaction on the intact enzyme) as the agent bringing about the cooperative conformational change in 12S.
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Xiaojing
X
Zheng
Rosa E
RE
Rivera-Hainaj
Yuangang
Y
Zheng
Marianne
M
Pusztai-Carey
Pamela R
PR
Hall
Vivien C
VC
Yee
Paul R
PR
Carey
D000214:Acyl Coenzyme A / Q000737:chemistry; D001665:Binding Sites; D019878:Carboxyl and Carbamoyl Transferases / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray; D008316:Malonyl Coenzyme A / Q000737:chemistry; D011424:Propionibacterium / Q000201:enzymology; D011487:Protein Conformation; D021122:Protein Subunits; D013059:Spectrum Analysis, Raman / Q000379:methods; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D021122:Protein Subunits; C015357:methylmalonyl-coenzyme A; C024343:butyryl-coenzyme A; C009061:propionyl-coenzyme A; D008316:Malonyl Coenzyme A; D019878:Carboxyl and Carbamoyl Transferases; C021580:Methylmalonyl-CoA carboxytransferase