Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

Biochem J. 1983 May 1; 211(2): 427–434.

PMCID: PMC1154375

H Kirsten and P Christen

Abstract

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.

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Selected References

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Alter, GM; Leussing, DL; Neurath, H; Vallee, BL. Kinetic properties of carboxypeptidase B in solutions and crystals. Biochemistry. 1977 Aug 9;16(16):3663–3668. [PubMed]
Amiconi, G; Bonaventura, C; Bonaventura, J; Antonini, E. Functional properties of normal and sickle cell hemoglobins in polyethylene glycol 6000. Biochim Biophys Acta. 1977 Dec 20;495(2):279–286. [PubMed]
Atha, DH; Ingham, KC. Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume. J Biol Chem. 1981 Dec 10;256(23):12108–12117. [PubMed]
Barra, D; Bossa, F; Doonan, S; Fahmy, HM; Hughes, GJ; Martini, F; Petruzzelli, R; Wittmann-Liebold, B. The cytosolic and mitochondrial aspartate aminotransferases from pig heart. A comparison of their primary structures, predicted secondary structures and some physical properties. Eur J Biochem. 1980 Jul;108(2):405–414. [PubMed]
Eichele, G; Karabelnik, D; Halonbrenner, R; Jansonius, JN; 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]
Eichele, G; Ford, GC; Glor, M; Jansonius, JN; 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. [PubMed]
Ford, GC; Eichele, G; Jansonius, JN. 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. [PubMed]
Gehring, H; Christen, P. Syncatalytic conformational changes in mitochondrial aspartate aminotransferases. Evidence from modification and demodification of Cys 166 in the enzyme from chicken and pig. J Biol Chem. 1978 May 10;253(9):3158–3163. [PubMed]
Gehring, H; Christen, P; Eichele, G; Glor, M; Jansonius, JN; Reimer, AS; Smit, JD; Thaller, C. Isolation, crystallization and preliminary crystallographic data of aspartate aminotransferase from chicken heart mitochondria. J Mol Biol. 1977 Sep;115(1):97–101. [PubMed]
Gehring, H; Rando, RR; Christen, P. Active-site labeling of aspartate aminotransferases by the beta,gamma-unsaturated amino acid vinylglycine. Biochemistry. 1977 Nov 1;16(22):4832–4836. [PubMed]
GINZBURG, BZ; KATCHALSKY, A. THE FRICTIONAL COEFFICIENTS OF THE FLOWS OF NON-ELECTROLYTES THROUGH ARTIFICIAL MEMBRANES. J Gen Physiol. 1963 Nov;47:403–418. [PubMed]
Ingham, KC. Polyethylene glycol in aqueous solution: solvent perturbation and gel filtration studies. Arch Biochem Biophys. 1977 Nov;184(1):59–68. [PubMed]
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. [PubMed]
Laidler, KJ; Bunting, PS. The kinetics of immobilized enzyme systems. Methods Enzymol. 1980;64:227–248. [PubMed]
Makinen, MW; Fink, AL. Reactivity and cryoenzymology of enzymes in the crystalline state. Annu Rev Biophys Bioeng. 1977;6:301–343. [PubMed]
McPherson, A., Jr Crystallization of proteins from polyethylene glycol. J Biol Chem. 1976 Oct 25;251(20):6300–6303. [PubMed]
NISONOFF, A; HENRY, SS; BARNES, FW., Jr Mechanisms in enzymatic transamination; variables in the spectrophotometric estimation of glutamate-aspartate kinetics. J Biol Chem. 1952 Dec;199(2):699–711. [PubMed]
Pfister, K; Kägi, JH; Christen, P. Syncatalytic conformational changes in aspartate aminotransferase determined by hydrogen-deuterium exchange. Proc Natl Acad Sci U S A. 1978 Jan;75(1):145–148. [PubMed]
Spilburg, CA; Bethune, JL; Valee, BL. Kinetic properties of crystalline enzymes. Carboxypeptidase A. Biochemistry. 1977 Mar 22;16(6):1142–1150. [PubMed]