Abstract
As a general rule protein concentration typical for structural studies differs considerably from that chosen for kinetic investigations. Consequently, structure-function relationships are often postulated without appropriate knowledge, whether the functional behaviour of the enzyme is the same in both protein concentration ranges. To deal with this question, substrate activation kinetics of two well-characterised yeast pyruvate decarboxylases, from Saccharomyces cerevisiae and from Kluyveromyces lactis, were analysed over the broad protein concentration range 2-2,000 μg/mL. Analytical ultracentrifugation and small-angle X-ray scattering were used to analyse the enzymes’ oligomer structure in aqueous solution. For the upper part of the concentration range the determined parameters, like catalytic activity, observed substrate activation rates, sedimentation coefficients and scattering parameters are independent on enzyme concentration changes. No indication of protein aggregation is detectable. However, significant changes occur at low enzyme concentration. The catalytically active tetramer dissociates progressively into dimers with comparable catalytic activity, but with significantly accelerated substrate activation.
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Abbreviations
- PDC:
-
Pyruvate decarboxylase (E.C.4.1.1.1)
- ThDP:
-
Thiamine diphosphate
- SAXS:
-
Small-angle X-ray solution scattering with synchrotron radiation
- ScPDC:
-
PDC from Saccharomyces cerevisiae
- KlPDC:
-
PDC from Kluyveromyces lactis
References
Schellenberger A, Hübner G, Neef H (1997) Methods Enzymol 279:131–146
Alvarez ME, Rosa AL, Temporini ED, Wolstenholme A, Panzetta G, Patrito L, Maccioni HJF (1993) Gene 130:253–258
Mücke U, König S, Hübner G (1995) Biol Chem Hoppe-Seyler 376:111–117
König S, Svergun DI, Volkov V, Feigin VA, Koch MHJ (1998) Biochemistry 37:5329–5334
König S, Svergun D, Koch MHJ, Hübner G, Schellenberger A (1992) Biochemistry 31:8726–8731
König S, Svergun D, Koch MHJ, Hübner G, Schellenberger A (1993) Eur Biophys J 22:185–194
König S, Wille G, Koch MHJ (2000) Hasylab Annual Report pp 319–320
Schütz A, Golbik R, Tittmann K, Svergun DI, Koch MHJ, Hübner G, König S (2003) Eur J Biochem 270:2322–2331
Bringer-Meyer S, Schimz KL, Sahm H (1986) Arch Microbiol 146:105–110
Davies DD (1967) Proc Biochem Soc 104:50P
Hübner G, Weidhase R, Schellenberger A (1978) Eur J Biochem 92:175–181
Lu G, Dobritzsch D, König S, Schneider G (1997) FEBS Lett 403:249–253
Lu G, Dobritzsch D, Baumann S, Schneider G, König S (2000) Eur J Biochem 267:861–868
Hübner G, Schellenberger A (1986) Biochem Intern 13:767–772
Dietrich A, König S (1997) FEBS Lett 400:42–44
Krieger F, Spinka M, Golbik R, Hübner G, König S (2002) Eur J Biochem 269:3259–3263
Alvarez FJ, Ermer J, Hübner G, Schellenberger A, Schowen RL (1991) J Am Chem Soc 113:8402–8409
König S, Hübner G, Schellenberger A (1990) Biomed Biochim Acta 49:465–471
Hübner G, König S, Schellenberger A, Koch M (1990) FEBS Lett 266:17–20
Dyda F, Furey W, Swaminathan S, Sax M, Farrenkopf B, Jordan F (1993) Biochemistry 32:6165–6170
Kutter S, Wille G, Relle S, Weiss MS, Hübner G, König S (2006) FEBS J 273:4199–4209
Wang J, Golbik R, Seliger B, Spinka M, Tittmann K, Hübner G, Jordan F (2001) Biochemistry 40:1755–1763
Sieber M, König S, Hübner G, Schellenberger A (1983) Biomed Biochim Acta 42:343–349
Laemmli UK (1970) Nature 227:680–685
Holzer H, Schultz G, Villar-Palasi C, Jüntgen-Sell J (1956) Biochem Z 337:331–344
Killenberg-Jabs M, Jabs A, Lilie H, Golbik R, Hübner G (2001) Eur J Biochem 268:1698–1704
Koch MHJ, Bordas J (1983) Nucl Instrum Methods 208:461–469
Gabriel A, Dauvergne F (1982) Nucl Instrum Methods 201:223–224
Boulin CJ, Kempf R, Gabriel A, Koch MHJ (1988) Nucl Instrum Methods A269:312–320
Svergun DI (1992) J Appl Crystallogr 25:495–503
Muller YA, Lindqvist Y, Furey W, Schulz GE, Jordan F, Schneider G (1993) Structure 1:95–103
Acknowledgements
The support of the European Community, Research Infrastructure Action under the FP6 “Structuring the European Research Area Specific Programme” to the EMBL Hamburg Outstation, Contract Number RII3-CT-2004-506008 is acknowledged. The authors thank the EMBL outstation for access to beam line X33 at the DORIS storage ring, DESY, Hamburg and PD Dr. Hauke Lilie for conducting the analytical ultracentrifugation runs.
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Kutter, S., Spinka, M., Koch, M.H.J. et al. The Influence of Protein Concentration on Oligomer Structure and Catalytic Function of Two Pyruvate Decarboxylases. Protein J 26, 585–591 (2007). https://doi.org/10.1007/s10930-007-9101-4
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DOI: https://doi.org/10.1007/s10930-007-9101-4