Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains.


Abstract

Glutamine synthetase (GS), Mr 622,000, from Escherichia coli contains 12 active sites formed at heterologous interfaces between subunits [Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., & Eisenberg, D. (1986) Nature (London) 323, 304-309]. Temperature-induced changes in UV spectra from 3 to 68 degrees C were reversible with the Mn2+- or Mg2+-enzyme at pH 7.0 (50 degrees C) in 100 mM KCl. No dissociation or aggregation of dodecamer occurred at high temperatures. The thermal transition involves the exposure of approximately 0.7 of the 2 Trp residues/subunit (by UV difference spectroscopy) and 2 of the 17 Tyr residues/subunit (change in exposure from 4.7 to 6.7 Tyr/subunit by second-derivative spectral analysis). Monitoring changes in Trp and Tyr exposure independently gives data that conform to a two-state model for partial unfolding with Tm values (where delta G unfolding = 0) differing by 2-3 degrees C at each level of [Mn2+] studied and with average delta HvH values of 80 and 94 kcal/mol, respectively. These observations suggest that two regions of the oligomeric structure unfold separately as independent transitions (random model). However, the data can be fit equally with a sequential model in which the Trp transition occurs first upon heating. By fitting with either model, Tm values increase from approximately 47 to approximately 54 degrees C with increasing free [Mn2+] from 3.6 to 49 microM but decrease from approximately 54 to approximately 43 degrees C by further increasing free [Mn2+] from 0.05 to 10 mM; such behavior indicates that the high-temperature form of the enzyme binds Mn2+ more weakly but has more binding sites than the native enzyme. The high-temperature Mn-enzyme form is somewhat less unfolded than is the catalytically inactive apoenzyme, which undergoes no further Trp or Tyr exposure on heating and therefore is assumed to be the high-temperature form of divalent cation-free GS. Adding substrates [ADP, L-Met-(SR)-sulfoximine, Gln, Gln + NH2OH, or Gln + ADP] to Mn.GS increased Tm to varying extents by preferential binding to the folded form. Indeed, the transition-state analogue complex GS.(Mn2.ADP.L-Met-(S)-sulfoximine phosphate)12 was stable in the folded form to at least 72 degrees C. Moreover, an Arrhenius plot for gamma-glutamyl transfer activity was linear from 4 to 72 degrees C with Ea = 18.3 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 3844 Extra Details: sequential model; transition-state ; transfer activity;,active-site structures; free energy of substrate binding

Submission Details

ID: rEBAGGjh

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:23 p.m.

Version: 1

Publication Details
Shrake A;Fisher MT;McFarland PJ;Ginsburg A,Biochemistry (1989) Partial unfolding of dodecameric glutamine synthetase from Escherichia coli: temperature-induced, reversible transitions of two domains. PMID:2571357
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