PubMed · 4229625
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W Straughn, R H Wagner. 1966-07-31. A simple method for preparing fibrinogen.. https://pubmed.ncbi.nlm.nih.gov/4229625/
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Kinetic as well as chemical modification studies have implicated the presence of an active site arginine in choline acetyltransferase, whose function is to stabilize coenzyme binding by interacting with the 3'-phosphate of the coenzyme A substrate. In order to identify this residue seven conserved arginines in rat choline acetyltransferase were converted to alanine by site-directed mutagenesis, and the properties of these mutants were compared with the wild type enzyme. Substitution of arginine 452 with alanine resulted in a 7-12-fold increase in the Km for both CoA and acetylcholine as well as kcat, with little change in the Km for dephospho-CoA. Product inhibition studies showed choline to be a competitive inhibitor with respect to acetylcholine, indicating R452A follows the same Theorell-Chance kinetic mechanism as the wild type enzyme. Similar results were obtained with R452Q and R452E, with the latter showing the largest changes in kinetic parameters. These findings are consistent with Arg-452 mutations increasing the rate constant, k5, for dissociation of the coenzyme from the enzyme. Direct evidence that arginine 452 is involved in coenzyme A binding was obtained by showing a 5-10-fold decrease in affinity of the R452A mutant for coenzyme A as determined by the ability to protect against phenylglyoxal inactivation as well as thermal inactivation.
To determine whether the source of carbon for the indirect pathway of hepatic glycogen synthesis differs between the periportal and pericentral zones, we studied seven 24-h-fasted conscious rats given a constant 2-h intraduodenal infusion of glucose, 40% labeled with [U-13C]glucose (99% 13C enriched), to raise and maintain plasma glucose concentration at approximately 10 mM. Glycogen, glutamate, aspartate, and alanine were selectively sampled from the periportal and pericentral zones of the liver by the dual-digitonin pulse technique and analyzed by 13C-NMR for positional isotopomer distribution and by gas chromatography-mass spectrometry for mass isotopomer distribution. Plasma glucose mass isotopomer distribution was determined from gas chromatography-mass spectrometry. The isotopomer distribution indicates that there was no significant difference between the zones with respect to 1) percent direct flux of glucose into the glycogen (periportal, 34 +/- 4; pericentral, 38 +/- 4), 2) extent of oxaloacetate/fumarate equilibration (periportal, 0.54 +/- .01;, pericentral, 0.53 +/- 0.01), 3) dilution of tracer in oxaloacetate (periportal, 0.64 +/- 0.07;, pericentral, 0.64 +/- 0.07), or 4) inflow of pyruvate versus tricarboxylic acid cycle flux (periportal, 0.70 +/- 0.20; pericentral, 0.68 +/- 0.16). Positional isotopomer populations, determined from the 13C-13C splitting in C3 and C4 of periportal and pericentral glycogen, were indistinguishable, indicating no significant differences in the source of the 3-carbon precursors for hepatic glycogen synthesis by the indirect pathway. In conclusion, glucose metabolism is the same in the periportal and pericentral zones with regard to 1) the relative flux of carbon via the direct/indirect pathways, 2) the source of the 3-carbon precursor used in the indirect pathway of glycogen synthesis, and 3) the flux of the 3-carbon precursors through the tricarboxylic acid cycle.
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