Structural determination of alginic acid and the effects of calcium binding as determined by high-field n.m.r.

The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex.

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.

Escherichia coli alpha-ketoglutarate dehydrogenase complex.

The alpha-ketoglutarate dehydrogenase complex from Escherichia coli catalyzes the hydrolysis of S-succinyl-CoA to succinate and CoASH. The reaction rate is dependent upon the presence of thiamin pyrophosphate and NADH, as well as the functional integrity of the alpha-lipoyl groups associated with the enzyme. The Km value for S-succinyl-CoA is 9.3 X 10(-5) M, and the maximum velocity is 0.02 mumol X min-1 X mg of protein-1 at pH 7 and 25 degrees C. This hydrolysis can be rationalized on the basis that succinyl thiamin pyrophosphate is generated under reductive succinylation conditions. Occasional diversion of succinyl thiamin pyrophosphate to hydrolysis produces succinate.