Crystal Versus Solution Structures of Thiamine Diphosphate-dependent Enzymes*

The quaternary structures of the thiamine diphosphate-dependent enzymes transketolase (EC 2.2.1.1; from Saccharomyces cerevisiae), pyruvate oxidase (EC1.2.3.3; from Lactobacillus plantarum), and pyruvate decarboxylase (EC 4.1.1.1; from Zymomonas mobilis and brewers' yeast, the latter in the native and pyruvamide-activated forms) were examined by synchrotron x-ray solution scattering. The experimental scattering data were compared with the curves calculated from the crystallographic models of these multisubunit enzymes. For all enzymes noted above, except the very compact pyruvate decarboxylase from Z. mobilis, there were significant differences between the experimental and calculated profiles. The changes in relative positions of the subunits in solution were determined by rigid body refinement. For pyruvate oxidase and transketolase, which have tight intersubunit contacts in the crystal, relatively small modifications of the quaternary structure (root mean square displacements of 0.23 and 0.27 nm, respectively) sufficed to fit the experimental data. For the enzymes with looser contacts (the native and activated forms of yeast pyruvate decarboxylase), large modifications of the crystallographic models (root mean square displacements of 0.58 and 1.53 nm, respectively) were required. A clear correlation was observed between the magnitude of the distortions induced by the crystal environment and the interfacial area between subunits.

[1]  D I Svergun,et al.  Protein hydration in solution: experimental observation by x-ray and neutron scattering. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Pletcher,et al.  Crystal and molecular structure of thiamine pyrophosphate hydrochloride. , 1972, Journal of the American Chemical Society.

[3]  C. Boulin,et al.  Data acquisition systems for linear and area X-ray detectors using delay line readout , 1988 .

[4]  H. Fromm,et al.  Nonaggregating mutant of recombinant human hexokinase I exhibits wild-type kinetics and rod-like conformations in solution. , 1999, Biochemistry.

[5]  D I Svergun,et al.  A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis. , 1998, Biophysical journal.

[6]  R. Schowen,et al.  Linkage of Catalysis and Regulation in Enzyme Action. Carbon Isotope Effects, Solvent Isotope Effects, and Proton Inventories for the Unregulated Pyruvate Decarboxylase of Zymomonas mobilis , 1995 .

[7]  G. Schneider,et al.  Analysis of an invariant cofactor-protein interaction in thiamin diphosphate-dependent enzymes by site-directed mutagenesis. Glutamic acid 418 in transketolase is essential for catalysis. , 1994, The Journal of biological chemistry.

[8]  M. Koch,et al.  An X‐ray solution scattering study of the cofactor and activator induced structural changes in yeast pyruvate decarboxylase (PDC) , 1990, FEBS letters.

[9]  G. Schulz,et al.  Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase , 1993, Science.

[10]  G J Williams,et al.  The Protein Data Bank: a computer-based archival file for macromolecular structures. , 1977, Journal of molecular biology.

[11]  G. Schulz,et al.  The refined structures of a stabilized mutant and of wild-type pyruvate oxidase from Lactobacillus plantarum. , 1994, Journal of molecular biology.

[12]  S. König,et al.  Substrate activation behaviour of pyruvate decarboxylase from Pisum sativum cv. Miko , 1997, FEBS letters.

[13]  G. Schneider,et al.  Three‐dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution. , 1992, The EMBO journal.

[14]  D. Svergun,et al.  Mathematical methods in small-angle scattering data analysis , 1991 .

[15]  A. V. Semenyuk,et al.  Small-angle-scattering-data treatment by the regularization method , 1988 .

[16]  G. Schneider,et al.  Refined structure of transketolase from Saccharomyces cerevisiae at 2.0 A resolution. , 1994, Journal of molecular biology.

[17]  C. Boulin,et al.  Data appraisal, evaluation and display for synchrotron radiation experiments: Hardware and software , 1986 .

[18]  R. Hjelm,et al.  Solution structures of dimeric kinesin and ncd motors. , 1999, Biochemistry.

[19]  W Furey,et al.  Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast Saccharomyces cerevisiae at 2.3 A resolution. , 1995, Journal of molecular biology.

[20]  A. Schellenberger Struktur und Wirkungsweise des aktiven Zentrums der Hefe‐Pyruvat‐Decarboxylase , 1967 .

[21]  F. Jordan,et al.  Structure-function relationships and flexible tetramer assembly in pyruvate decarboxylase revealed by analysis of crystal structures. , 1998, Biochimica et biophysica acta.

[22]  B. Fedorov,et al.  Improved technique for calculating X‐ray scattering intensity of biopolymers in solution: Evaluation of the form, volume, and surface of a particle , 1983 .

[23]  Akira Matsunawa,et al.  The simulation of front keyhole wall dynamics during laser welding , 1997 .

[24]  D. I. Svergun,et al.  Structure Analysis by Small-Angle X-Ray and Neutron Scattering , 1987 .

[25]  D. I. Svergun,et al.  Solution scattering from biopolymers: advanced contrast‐variation data analysis , 1994 .

[26]  D. Svergun,et al.  Small-angle X-ray solution-scattering studies on ligand-induced subunit interactions of the thiamine diphosphate dependent enzyme pyruvate decarboxylase from different organisms. , 1998, Biochemistry.

[27]  D. Svergun,et al.  Synchrotron radiation solution X-ray scattering study of the pH dependence of the quaternary structure of yeast pyruvate decarboxylase. , 1992, Biochemistry.

[28]  R. Schowen,et al.  The linkage of catalysis and regulation in enzyme action. Solvent isotope effects as probes of protonic sites in the yeast pyruvate decarboxylase mechanism , 1995 .

[29]  D I Svergun,et al.  Solution structure of the ternary complex between aminoacyl-tRNA, elongation factor Tu, and guanosine triphosphate. , 1998, Biochemistry.

[30]  J. G. Grossmann,et al.  X-ray scattering using synchrotron radiation shows nitrite reductase from Achromobacter xylosoxidans to be a trimer in solution. , 1993, Biochemistry.

[31]  D I Svergun,et al.  Large differences are observed between the crystal and solution quaternary structures of allosteric aspartate transcarbamylase in the R state , 1997, Proteins.

[32]  J. Bordas,et al.  X-ray diffraction and scattering on disordered systems using synchrotron radiation , 1983 .

[33]  Gunter Schneider,et al.  High Resolution Crystal Structure of Pyruvate Decarboxylase from Zymomonas mobilis , 1998, The Journal of Biological Chemistry.

[34]  W Furey,et al.  Catalytic centers in the thiamin diphosphate dependent enzyme pyruvate decarboxylase at 2.4-A resolution. , 1993, Biochemistry.

[35]  F. Dauvergne,et al.  The localisation method used at EMBL , 1982 .

[36]  G. Hübner,et al.  The mechanism of substrate activation of pyruvate decarboxylase: a first approach. , 1978, European journal of biochemistry.

[37]  J. Ninio,et al.  Comparative small-angle x-ray scattering studies on unacylated, acylated and cross-linked Escherichia coli transfer RNA I Val . , 1972, Journal of molecular biology.