Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase

Miriam Gulotta, Linlin Qiu, Ruel Desamero, Jörg Rösgen, D. Wayne Bolen, Robert Callender

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

During cell volume regulation, intracellular concentration changes occur in both inorganic and organic osmolytes in order to balance the extracellular osmotic stress and maintain cell volume homeostasis. Generally, salt and urea increase the Km's of enzymes and trimethylamine N-oxide (TMAO) counteracts these effects by decreasing Km's. The hypothesis to account for these effects is that urea and salt shift the native state ensemble of the enzyme toward conformers that are substrate-binding incompetent (BI), while TMAO shifts the ensemble toward binding competent (BC) species. Km's are often complex assemblies of rate constants involving several elementary steps in catalysis, so to better understand osmolyte effects we have focused on a single elementary event, substrate binding. We test the conformational shift hypothesis by evaluating the effects of salt, urea, and TMAO on the mechanism of binding glycerol 3-phosphate, a substrate analogue, to yeast triosephosphate isomerase. Temperature-jump kinetic measurements promote a mechanism consistent with osmolyte-induced shifts in the [BI]/[BC] ratio of enzyme conformers. Importantly, salt significantly affects the binding constant through its effect on the activity coefficients of substrate, enzyme, and enzyme-substrate complex, and it is likely that TMAO and urea affect activity coefficients as well. Results indicate that the conformational shift hypothesis alone does not account for the effects of osmolytes on Km's.

Original languageEnglish (US)
Pages (from-to)10055-10062
Number of pages8
JournalBiochemistry
Volume46
Issue number35
DOIs
StatePublished - Sep 4 2007

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Triose-Phosphate Isomerase
Cell Size
Urea
Salts
Substrates
Enzymes
Activity coefficients
Osmotic Pressure
Catalysis
Yeast
Rate constants
Homeostasis
Yeasts
alpha-glycerophosphoric acid
Kinetics
Temperature
trimethyloxamine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase. / Gulotta, Miriam; Qiu, Linlin; Desamero, Ruel; Rösgen, Jörg; Bolen, D. Wayne; Callender, Robert.

In: Biochemistry, Vol. 46, No. 35, 04.09.2007, p. 10055-10062.

Research output: Contribution to journalArticle

Gulotta, Miriam ; Qiu, Linlin ; Desamero, Ruel ; Rösgen, Jörg ; Bolen, D. Wayne ; Callender, Robert. / Effects of cell volume regulating osmolytes on glycerol 3-phosphate binding to triosephosphate isomerase. In: Biochemistry. 2007 ; Vol. 46, No. 35. pp. 10055-10062.
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