Insight into the Carboxyl Transferase Domain Mechanism of Pyruvate Carboxylase from \u3cem\u3eRhizobium etli\u3c/em\u3e

The effects of mutations in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase have been determined for the forward reaction to form oxaloacetate, the reverse reaction to form MgATP, the oxamate-induced decarboxylation of oxaloacetate, the phosphorylation of MgADP by carbamoyl phosphate, and the bicarbonate-dependent ATPase reaction. Additional studies with these mutants examined the effect of pyruvate and oxamate on the reactions of the biotin carboxylase domain. From these mutagenic studies, putative roles for catalytically relevant active site residues were assigned and a more accurate description of the mechanism of the carboxyl transferase domain is presented. The T882A mutant showed no catalytic activity for reactions involving the carboxyl transferase domain but surprisingly showed 7- and 3.5-fold increases in activity, as compared to that of the wild-type enzyme, for the ADP phosphorylation and bicarbonate-dependent ATPase reactions, respectively. Furthermore, the partial inhibition of the T882A-catalyzed BC domain reactions by oxamate and pyruvate further supports the critical role of Thr882 in the proton transfer between biotin and pyruvate in the carboxyl transferase domain. The catalytic mechanism appears to involve the decarboxylation of carboxybiotin and removal of a proton from Thr882 by the resulting biotin enolate with either a concerted or subsequent transfer of a proton from pyruvate to Thr882. The resulting enolpyruvate then reacts with CO2 to form oxaloacetate and complete the reaction

1861 - The Pathfinder

Document citation: The War of the Rebellion: Official Records of the Union and Confederate Armies (Washington: Government Printing Office, 1880-1901), series 1, volume 3, 467.https://scholarworks.uni.edu/nhomefront/1009/thumbnail.jp

Probing the Catalytic Roles of Arg548 and Gln552 in the Carboxyl Transferase Domain of the \u3cem\u3eRhizobium etli\u3c/em\u3e Pyruvate Carboxylase by Site-directed Mutagenesis

The roles of Arg548 and Gln552 residues in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase were investigated using site-directed mutagenesis. Mutation of Arg548 to alanine or glutamine resulted in the destabilization of the quaternary structure of the enzyme, suggesting that this residue has a structural role. Mutations R548K, Q552N, and Q552A resulted in a loss of the ability to catalyze pyruvate carboxylation, biotin-dependent decarboxylation of oxaloacetate, and the exchange of protons between pyruvate and water. These mutants retained the ability to catalyze reactions that occur at the active site of the biotin carboxylase domain, i.e., bicarbonate-dependent ATP cleavage and ADP phosphorylation by carbamoyl phosphate. The effects of oxamate on the catalysis in the biotin carboxylase domain by the R548K and Q552N mutants were similar to those on the catalysis of reactions by the wild-type enzyme. However, the presence of oxamate had no effect on the reactions catalyzed by the Q552A mutant. We propose that Arg548 and Gln552 facilitate the binding of pyruvate and the subsequent transfer of protons between pyruvate and biotin in the partial reaction catalyzed in the active site of the carboxyl transferase domain of Rhizobium etli pyruvate carboxylase

Preparation of corn for yearling brood sows

Ear corn and shelled corn proved more efficient than ground ear corn or corn meal for wintering brood sows. This was the result obtained where four groups of 10 sows each received salt (self-fed), and three-fifths of a pound per head daily of a mixture of linseed oilmeal, tankage and wheat middlings, in addition to one of the following: ear corn, shelled corn, corn and cob meal, or ground corn. The differences in gains from the various kinds of corn preparation were not great. Hence, the objection to grinding the corn lies in the cost. Ground shelled corn proved especially costly

Dried buttermilk for growing and fattening pigs

The objects of this experiment were to determine the comparative value and efficiency of meat meal tankage, dried buttermilk and various combinations of these feeds on health, gains, feed requirements, water consumption, time required to reach 225 pounds average weight and other factors, when these protein feeds were self-fed along with shelled corn to growing and fattening pigs in dry lots. The pigs used in this test were very uniform in age, weight and breeding. They were all sired by the same purebred Hampshire boar and were out of either related purebred or very high grade Hampshire sows. The pigs were selected from four litters

Avidin as a probe of the conformational changes induced in pyruvate carboxylase by acetyl-CoA and pyruvate

AbstractSheep liver pyruvate carboxylase was mixed with avidin at a molar ratio of 1:1 in the presence of various combinations of the components of the assay systems required for either the acetyl-CoA-dependent or the acetyl-CoA-independent activity and negatively stained samples were examined by electron microscopy. Significant numbers of chain-like polymers of enzyme-avidin complexes were evident only when acetyl-CoA or high levels of pyruvate were present in the media. Similar results were also obtained for chicken liver pyruvate carboxylase despite this enzyme's almost complete lack of acetyl-CoA-independent activity. Thus, although acetyl-CoA and high concentrations of pyruvate may induce pyruvate carboxylase to adopt a ‘tight’ tetrahedron-like conformation which can interact with avidin to form chains, this structural change alone does not result in an enzymic form that is maximally active. This suggests that the allosteric activation of pyruvate carboxylase by acetyl-CoA is attributable, at least in part to more subtle conformational changes, especially in the case of the chicken enzyme

What Explains the Incidence of the Use of a Common Sediment Control on Lots with Houses Under Construction?

To analyze compliance with one aspect of the regulation of stormwater discharge, we estimate a random-utility model of the probability that a builder uses a silt fence to control sediments on a lot with a house under construction in an urbanizing county of South Carolina. The probability increases if the builder is responsible to the subdivision’s developer or if a homeowners association exists. The probability also increases as the cost to install a silt fence decreases or the number of houses under construction per built house in a subdivision increases. The results can help county officials target inspection to improve compliance.compliance with regulation, erosion and sediment control, filter fabric, management of stormwater runoff, random-utility model, silt fence, storm water pollution prevention plan, Agribusiness, Community/Rural/Urban Development, Demand and Price Analysis, Environmental Economics and Policy, Industrial Organization, Land Economics/Use, Q01, Q24, Q53, Q58,

A broadband study of the 13 August 1978 Santa Barbara earthquake

The Santa Barbara earthquake of 13 August 1978, provides an opportunity to perform a broadband investigation of body waves for a well-recorded, moderate size (M_L = 5.1) event. The long- and short-period teleseismic body waves are modeled in the time domain to construct a source time function which is consistent in the period range of 1 to 20 sec. The long-period records indicate an overall duration of 6 sec while the short-period records reveal the fine-scale character of the slip history consisting of two sharp pulses separated by about 1 sec. The source mechanism determined from this analysis is a moderately dipping (30°NE) thrust with significant left-lateral slip. The moment was determined to be 1.1 × 10^(25) dyne-cm. The earthquake was also reasonably well recorded on accelerographs in the near-field. The modeling of the strong motion displacements was a two step procedure: (1) the displacements were modeled alone, and (2) in an attempt to achieve consistency between the local and far-field time functions, the qualitative features of the teleseismic short-period time function were used to predict the displacements. If the two sources in the short-period time function are allowed to have different mechanisms, the displacements can be modeled quite well. This suggests that the overall faulting process was rough, and the multiple source character suggested at high frequencies is due to high-stress drop asperities. The two sources are modeled as asperities separated by 1.5 km; the first source has a mechanism consistent with the teleseismic solution while the second source is more steeply dipping. The total moment determined from the strong motion data is 3.5 × 10^(24) dyne-cm or one-third the long-period moment. This is consistent with other recent studies which suggest that the high-frequency strong ground motion is controlled by the distribution of asperities even though the sum of their moments may be small compared to the overall moment. This study also shows the importance of teleseismic short periods in predicting the local displacements

The evolution of the sponge feeding habit in the caddisfly genus Ceraclea (Trichoptera, Leptoceridae)

A phylogeny is proposed for 11 species of Ceraclea caddisflies which feed on freshwater sponge during the larval stage of their life cycle. Based on this evolutionary scheme, further probable sponge feeders are postulated. Some morphological differences of the sponge feeders are described. The obligate sponge feeders are morphologically and ecologically different from other species of the genus which may feed on sponge when it is available or when other food sources are scarce, and from the species of Ceraclea which appear never to eat sponge