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. 1983 Apr;80(7):1807–1810. doi: 10.1073/pnas.80.7.1807

Crystalline aspartate aminotransferase: lattice-induced functional asymmetry of the two subunits.

H Kirsten, H Gehring, P Christen
PMCID: PMC393698  PMID: 6572940

Abstract

The enzymic activity of crystalline mitochondrial aspartate aminotransferase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) was determined in suspensions of noncrosslinked microcrystals in 30% (wt/vol) polyethylene glycol. The crystals (average dimensions, 22 x 5 x 0.8 micron) were small enough to preclude diffusional rate limitation. They had the same habit as the triclinic crystals used for the determination of the spatial structure of the enzyme by x-ray crystallographic analysis [Ford, G. C., Eichele, G., and Jansonius, J. N. (1980) Proc. Natl. Acad. Sci. USA 77, 2559-2563]. Determination of the Michaelis-Menten parameters showed that the packing of the enzyme dimer into the crystal lattice not only decreases its activity but also induces a functional nonequivalence of the two subunits that behave identically in solution. The crystalline enzyme possesses a high-affinity subunit with Km values similar to those of the enzyme in solution (K'm = 0.5 mM for aspartate and 1.2 mM for 2-oxoglutarate) and a low-affinity subunit (K'm = 5.5 mM and 14.5 mM, respectively). The catalytic activity of the high-affinity subunit is 3% and that of the low-affinity subunit is 15% of the activity of the enzyme in solution. The functional asymmetry of the crystalline enzyme dimer could also be demonstrated by selective mechanism-based modification of either type of active sites. In view of the apparently identical conformation of the two subunits in the crystalline enzyme, its decreased catalytic efficiency and its functional asymmetry likely are due to constraints exerted by the crystal lattice on the conformational adaptability of the two subunits. In triclinic crystals the two subunits of the enzyme dimer have dissimilar lattice contacts.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alter G. M., Leussing D. L., Neurath H., Vallee B. L. Kinetic properties of carboxypeptidase B in solutions and crystals. Biochemistry. 1977 Aug 9;16(16):3663–3668. doi: 10.1021/bi00635a024. [DOI] [PubMed] [Google Scholar]
  2. Boettcher B., Martinez-Carrion M. Hybridization of glutamate aspartate transaminase. Investigation of subunit interaction. Biochemistry. 1975 Oct 7;14(20):4528–4531. doi: 10.1021/bi00691a030. [DOI] [PubMed] [Google Scholar]
  3. Boettcher B., Martinez-Carrion M. Itemizing enzyme ligand interactions in native and and half-active hybrid aspartate transaminase to probe site-site relationships. Biochemistry. 1976 Dec 14;15(25):5657–5664. doi: 10.1021/bi00670a035. [DOI] [PubMed] [Google Scholar]
  4. Chance B., Ravilly A. Reactivity of crystalline ferrihemoglobin towards azide. J Mol Biol. 1966 Oct 28;21(1):195–198. doi: 10.1016/0022-2836(66)90087-8. [DOI] [PubMed] [Google Scholar]
  5. Eichele G., Ford G. C., Glor M., Jansonius J. N., Mavrides C., Christen P. The three-dimensional structure of mitochondrial aspartate aminotransferase at 4.5 A resolution. J Mol Biol. 1979 Sep 5;133(1):161–180. doi: 10.1016/0022-2836(79)90255-9. [DOI] [PubMed] [Google Scholar]
  6. Eichele G., Karabelnik D., Halonbrenner R., Jansonius J. N., Christen P. Catalytic activity in crystals of mitochondrial aspartate aminotransferase as detected by microspectrophotometry. J Biol Chem. 1978 Aug 10;253(15):5239–5242. [PubMed] [Google Scholar]
  7. Feldman H. A. Mathematical theory of complex ligand-binding systems of equilibrium: some methods for parameter fitting. Anal Biochem. 1972 Aug;48(2):317–338. doi: 10.1016/0003-2697(72)90084-x. [DOI] [PubMed] [Google Scholar]
  8. Ford G. C., Eichele G., Jansonius J. N. Three-dimensional structure of a pyridoxal-phosphate-dependent enzyme, mitochondrial aspartate aminotransferase. Proc Natl Acad Sci U S A. 1980 May;77(5):2559–2563. doi: 10.1073/pnas.77.5.2559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. GINZBURG B. Z., KATCHALSKY A. THE FRICTIONAL COEFFICIENTS OF THE FLOWS OF NON-ELECTROLYTES THROUGH ARTIFICIAL MEMBRANES. J Gen Physiol. 1963 Nov;47:403–418. doi: 10.1085/jgp.47.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Gehring H., Christen P., Eichele G., Glor M., Jansonius J. N., Reimer A. S., Smit J. D., Thaller C. Isolation, crystallization and preliminary crystallographic data of aspartate aminotransferase from chicken heart mitochondria. J Mol Biol. 1977 Sep;115(1):97–101. doi: 10.1016/0022-2836(77)90249-2. [DOI] [PubMed] [Google Scholar]
  11. HUGHES R. C., JENKINS W. T., FISCHER E. H. The site of binding of pyridoxal-5'-phosphate to heart glutamic-aspartic transaminase. Proc Natl Acad Sci U S A. 1962 Sep 15;48:1615–1618. doi: 10.1073/pnas.48.9.1615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ingham K. C. Polyethylene glycol in aqueous solution: solvent perturbation and gel filtration studies. Arch Biochem Biophys. 1977 Nov;184(1):59–68. doi: 10.1016/0003-9861(77)90326-5. [DOI] [PubMed] [Google Scholar]
  13. KARMEN A. A note on the spectrometric assay of glutamic-oxalacetic transaminase in human blood serum. J Clin Invest. 1955 Jan;34(1):131–133. [PubMed] [Google Scholar]
  14. Katchalski E., Silman I., Goldman R. Effect of the microenvironment on the mode of action of immobilized enzymes. Adv Enzymol Relat Areas Mol Biol. 1971;34:445–536. doi: 10.1002/9780470122792.ch7. [DOI] [PubMed] [Google Scholar]
  15. Laidler K. J., Bunting P. S. The kinetics of immobilized enzyme systems. Methods Enzymol. 1980;64:227–248. doi: 10.1016/s0076-6879(80)64011-7. [DOI] [PubMed] [Google Scholar]
  16. Lee Y. H., Churchich J. E. Mitochondrial aspartate aminotransferase-independent function of the catalytic binding sites. J Biol Chem. 1975 Jul 25;250(14):5604–5608. [PubMed] [Google Scholar]
  17. Makinen M. W., Fink A. L. Reactivity and cryoenzymology of enzymes in the crystalline state. Annu Rev Biophys Bioeng. 1977;6:301–343. doi: 10.1146/annurev.bb.06.060177.001505. [DOI] [PubMed] [Google Scholar]
  18. McPherson A., Jr Crystallization of proteins from polyethylene glycol. J Biol Chem. 1976 Oct 25;251(20):6300–6303. [PubMed] [Google Scholar]
  19. Metzler C. M., Metzler D. E., Martin D. S., Newman R., Arnone A., Rogers P. Crystalline enzyme.substrate complexes of asparate aminotransferase. J Biol Chem. 1978 Aug 10;253(15):5251–5254. [PubMed] [Google Scholar]
  20. Mozzarelli A., Ottonello S., Rossi G. L., Fasella P. Catalytic activity of aspartate aminotransferase in the crystal. Equilibrium and kinetic analysis. Eur J Biochem. 1979 Jul;98(1):173–179. doi: 10.1111/j.1432-1033.1979.tb13174.x. [DOI] [PubMed] [Google Scholar]
  21. QUIOCHO F. A., RICHARDS F. M. INTERMOLECULAR CROSS LINKING OF A PROTEIN IN THE CRYSTALLINE STATE: CARBOXYPEPTIDASE-A. Proc Natl Acad Sci U S A. 1964 Sep;52:833–839. doi: 10.1073/pnas.52.3.833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schlegel H., Christen P. Cytosolic aspartate aminotransferase. Studies on subunit interactions with the apo/holo hybrid dimer. Biochim Biophys Acta. 1978 Jan 25;532(1):6–16. doi: 10.1016/0005-2795(78)90442-7. [DOI] [PubMed] [Google Scholar]
  23. Schlegel H., Christen P. The apo-holo hybrid of cytosolic aspartate aminotransferase, preparation and studies on subunit interactions. Biochem Biophys Res Commun. 1974 Nov 6;61(1):117–123. doi: 10.1016/0006-291x(74)90542-7. [DOI] [PubMed] [Google Scholar]
  24. Schlegel H., Zaoralek P. E., Christen P. Aspartate aminotransferase. Determination of the active site occupancy pattern indicates independent transamination of the two subunits. J Biol Chem. 1977 Aug 25;252(16):5835–5838. [PubMed] [Google Scholar]
  25. Spilburg C. A., Bethune J. L., Valee B. L. Kinetic properties of crystalline enzymes. Carboxypeptidase A. Biochemistry. 1977 Mar 22;16(6):1142–1150. doi: 10.1021/bi00625a018. [DOI] [PubMed] [Google Scholar]
  26. Spilburg C. A., Bethune J. L., Vallee B. L. The physical state dependence of carboxypeptidase Aalpha and Agamma kinetics. Proc Natl Acad Sci U S A. 1974 Oct;71(10):3922–3926. doi: 10.1073/pnas.71.10.3922. [DOI] [PMC free article] [PubMed] [Google Scholar]

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