NMR solution structure of the theta subunit of DNA polymerase III from Escherichia coli.

The catalytic core of Escherichia coli DNA polymerase III contains three tightly associated subunits (alpha, epsilon, and theta). The theta subunit is the smallest, but the least understood of the three. As a first step in a program aimed at understanding its function, the structure of the theta subunit has been determined by triple-resonance multidimensional NMR spectroscopy. Although only a small protein, theta was difficult to assign fully because approximately one-third of the protein is unstructured, and some sections of the remaining structured parts undergo intermediate intramolecular exchange. The secondary structure was deduced from the characteristic nuclear Overhauser effect patterns, the 3J(HN alpha) coupling constants and the consensus chemical shift index. The C-terminal third of the protein, which has many charged and hydrophilic amino acid residues, has no well-defined secondary structure and exists in a highly dynamic state. The N-terminal two-thirds has three helical segments (Gln10-Asp19, Glu38-Glu43, and His47-Glu54), one short extended segment (Pro34-Ala37), and a long loop (Ala20-Glu29), of which part may undergo intermediate conformational exchange. Solution of the three-dimensional structure by NMR techniques revealed that the helices fold in such a way that the surface of theta is bipolar, with one face of the protein containing most of the acidic residues and the other face containing most of the long chain basic residues. Preliminary chemical shift mapping experiments with a domain of the epsilon subunit have identified a loop region (Ala20-Glu29) in theta as the site of association with epsilon.

13C n.m.r. isotopomer and computer-simulation studies of the non-oxidative pentose phosphate pathway of human erythrocytes.

13C double-quantum filtered correlation spectroscopy (DQF-COSY) provides a novel method for the detection of reactions involving carbon-bond scissions. We report the use of this technique to investigate isotopic exchange reactions of the non-oxidative pentose phosphate pathway in human erythrocytes. These exchange reactions resulted in the formation of a range of isotopic isomers (isotopomers) of glucose 6-phosphate after incubation of a mixture of universally 13C-labelled and unlabelled glucose 6-phosphate with fructose 1,6-bisphosphate and haemolysates. These isotopomers were detected in the coupling patterns of cross-peaks within the DQF-COSY spectrum of the deproteinized sample. A computer model which fully describes the reactions of the non-oxidative pentose phosphate pathway in human erythrocytes has previously been constructed and tested with 31P n.m.r. time-course data in our laboratory. This model was refined using 13C n.m.r. time-course data and extended to include the range of isotopomers which may be formed experimentally by the reactions of the non-oxidative pentose phosphate pathway. The isotopomer ratios obtained experimentally from the DQF-COSY spectrum were consistent with simulations generated by this model.

High control coefficient of transketolase in the nonoxidative pentose phosphate pathway of human erythrocytes: NMR, antibody, and computer simulation studies.

The degree of control exerted by transketolase over metabolite flux in the nonoxidative pentose phosphate pathway in human erythrocytes was investigated using transketolase antiserum to modulate the activity of that enzyme. 31P NMR enabled the simultaneous measurement of the levels of pentose phosphate pathway metabolites following incubation of hemolysates with ribose 5-phosphate. The variations in metabolic flux which occurred as the transketolase activity of hemolysate samples was altered indicated that a high degree of control was exerted by transketolase. Investigations using transaldolase-depleted hemolysates showed that transaldolase exhibits a lesser degree of control over pathway flux. Experimental data were compared with simulations generated by a computer model encompassing the reactions of the classical nonoxidative pentose phosphate pathway. The sensitivity coefficients (also called "control strengths" or "flux-control coefficients") calculated from the computer simulations were 0.74 and 0.03 for transketolase and transaldolase, respectively.

13C and 31P NMR studies of the pentose phosphate pathway in human erythrocytes.

31P NMR was used to monitor the concentrations of some of the intermediates of the non-oxidative pentose phosphate pathway (PPP) during the dissimilation of inosine and phosphate in dilute haemolysates. The temperature dependence of the ketone/hydrate ratio in dihydroxyacetone phosphate and the relative proportions of the isomers of sedoheptulose 1,7-bisphosphate were measured. The 31P NMR time courses were compared with the simulations obtained with a computer model of the modified F-type PPP.

Computer simulation of the pentose-phosphate pathway and associated metabolism used in conjunction with NMR experimental data from human erythrocytes.

A computer-based model of the metabolism of sugar phosphates by human erythrocytes has been developed to assist in the understanding of the biochemical transformations occurring in the pentose phosphate pathway. These transformations are reflected in the changes, with time, of the relative intensities of the metabolite peaks apparent in 1H, 13C and 31P NMR spectra. The deterministic model consists of 79 reactions interconnected in a defined structure and characterized by 155 rate constants. It also includes 17 different enzymes, 69 enzyme forms, 32 metabolites, and initial value of time and concentration of each of the reactants. The differential equations describing the time-dependence of the concentrations of the reactants are generated and then solved by using the computer program BIOSSIM, which is designed to solve arrays of "stiff" differential equations. We synthesized [1-13C]D-ribose 5-phosphate and used 13C and 31P NMR to monitor its transformation into various intermediates of the pentose phosphate pathway, after the addition of diluted haemolysates which had previously been depleted of nicotinamide- and adenine-nucleotides. The concentrations of several of the reactants were able to be quantified, while other peaks in both the 13C and 31P spectra are yet to be assigned with confidence. There was reasonable qualitative agreement between some aspects of the computer simulation of the proposed metabolic system and the experimental data.