+ Translate

Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: Selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea

, : Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: Selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea. Biochemistry 32(16): 4156-4167

Structural aspects requisite for allosteric function in the regulatory chain of aspartate transcarbamoylase were explored by site-specific amino acid insertion or substitution within the zinc domain in the region of contact between the catalytic and regulatory chains. Amino acid substitution at two positions yielded enzymes which retained a maximum velocity similar to that of the wild-type enzyme but responded differently from the native enzyme in the presence of regulatory nucleoside triphosphates. A change of zinc coordinate amino acid C109 to histidine and a change of El19 to aspartic acid resulted in enzymes which demonstrated synergistic inhibition by CTP and UTP but not inhibition by CTP in either phosphate buffer or a morpholino-based tripartate (TP) buffer at pH 7. At pH 8.3, where there is a higher proportion of T-state conformers in the native enzyme, the mutants diverged from their similar kinetic behavior. C109H remained an enzyme which was not inhibited by CTP but was still inhibited by CTP+UTP. E119D was inhibited by both CTP and CTP+UTP. Activation of the mutants by ATP was found to vary either with pH or with phosphate as a buffer component. C109H was activated by ATP in phosphate, while in TP at either pH 7 or 8.3 its activation by ATP was diminished or absent. E119D was activated by ATP in phosphate at pH 7 or in TP at pH 8.3, but not in TP at pH 7. In TP at pH 7, where neither mutant was activated by ATP, the S-0.5 values and Hill coefficients of the unliganded mutant enzymes resembled those of the ATP-liganded wild-type enzyme. While neither mutation would be predicted to alter the net charge of the holoenzyme, differences in the isolectric point of the mutants were observed if phosphate was present. This result suggests that the isoelectric point of aspartate transcarbamoylase is conformationally dependent and that the mutants exist in an altered conformation. In addition, the stabilities of both mutant holoenzymes were reduced substantially from those of the wild-type enzyme in 4 M urea. C109H was more stable at pH 8.25 in a Tris buffer; E119D was more stable at pH 7 in the phosphate buffer. Potential effects of these mutations on the active site chemistry and geometry are discussed.

Order PDF Full Text


Click here to order any other PDF Full Text

Accession: 009422455

PMID: 8476846

DOI: 10.1021/bi00067a002

Download PDF Full Text: Site-directed alterations to the geometry of the aspartate transcarbamoylase zinc domain: Selective alteration to regulation by heterotropic ligands, isoelectric point, and stability in urea

Submit PDF Full Text

No spam - Every submission is manually reviewed

Due to poor quality, we do not accept files from Researchgate

Submitted PDF Full Texts will always be free for everyone
(We only charge for PDFs that we need to acquire)

Select a PDF file:

Other references

Vincent,B.; FotiniKoutroumpa,V.A.; Roux-Morabito,G.; Gevar,J.; Martin,C.; Lieutier,F., 2008: Occurrence of Bursaphelenchus mucronatus in France and association with Monochamus galloprovincialis. As a consequence of the recent introduction of the pine wood nematode <i>Bursaphelenchus xylophilus</i> in Portugal, nematodes of the genus <i>Bursaphelenchus</i> in various French pine forests, in trees attacked by <i&g...

Mayer, J.W.; Dehoratius, R.J.; Messner, R.P., 1976: Serratia marcescens caused arthritis with negative and positive birefringent crystals. An unusual case of arthritis caused by S. marcescens, with positive and negative birefringent crystals in the same inflammatory synovial fluid was encountered. This combination of events is most likely to occur in men over 40 yr old who have a pre...

Han, C.; McGue, M.K.; Iacono, W.G., 2000: Lifetime tobacco, alcohol and other substance use in adolescent Minnesota twins: univariate and multivariate behavioral genetic analyses. We sought to estimate the contribution of genetic and environmental factors to adolescent tobacco, alcohol and other substance use. The sample consisted of 327 monozygotic and 174 like-sex dizygotic twin pairs born in Minnesota and aged 17-18 year...

Sadeghi, N.G.; Pariyadath, V.; Apte, S.; Eagleman, D.M.; Cook, E.P., 2012: Neural correlates of subsecond time distortion in the middle temporal area of visual cortex. How does the brain represent the passage of time at the subsecond scale? Although different conceptual models for time perception have been proposed, its neurophysiological basis remains unknown. We took advantage of a visual duration illusion pro...

Chaturvedi, A. N., 1973: General standard volume tables for Deodar (Cedrus deodara). Presents tables for merchantable timber volumes and smallwood volumes of C. deodara, based on data from Himachal Pradesh, Jammu and Kashmir, and Uttar Pradesh.

Croci, L.; Draisci, R.; Lucentini, L.; Cozzi, L.; Giannetti, L.; Toti, L.; Stacchini, A., 1995: Influence of the extraction procedure on recovery of okadaic acid from experimentally contaminated mussels. Hepatopancreas samples from mussels (Mytilus galloprovincialis) experimentally contaminated with okadaic acid were analysed with Yasumoto's mouse bioassay and HPLC. A likely effect of some components of the hepatopancreas on the results (matr...

Patterson, W.C.; Siegel, H.S.; Drury, L.N., 1973: Downgrading associated with growing broilers in cages. In 2 trials, White Plymouth Rocks were reared in plastic cages (323 cm2/bird) or in floor pens (656 cm2/bird) under 2 temp. regimes in 2 windowless rooms. The temp. in one room was a constant 35.0 deg C, while in the other it was normal, decreasin...

Hermann, G.; Abdelwahab, I.F.; Kenan, S.; Lewis, M.M.; Klein, M.J., 1993: Case report 795. High-grade surface osteosarcoma of the radius. We described a case of high-grade surface sarcoma of the radius in a 14-year-old boy. The lesion appeared radiologically benign and was confined to the surface. The tumor was resected. At the time of biopsy both lungs showed diffuse metastases. Fo...

Zhebrovskaia, Vu, 1977: Conversion of proteins in the stomachs of early-weaned piglets with varied consistency of feed. Biulleten' nauchnykh rabot: 2) 18-19

Srivastava, V.; Viswanathaswamy, A.H.M.; Mohan, G., 2010: Determination of the antiulcer properties of sodium cromoglycate in pylorus-ligated albino rats. To study the ulcer protective property of sodium cromoglycate in pylorusligated rats and the biochemical role in ulcer protection by various biochemical tests. The ulcer protective effect of sodium cromoglycate was studied using a Pyloric Ligation...