Yeast hexokinase has been poorly characterized in regard with its stability. In the present study, various spectroscopic techniques were employed to investigate thermal stability of the monomeric form of yeast hexokinase B (YHB). The enzyme underwent a conformational transition with a Tm of about 41.9 °C. The structural transition proved to be significantly reversible below 55 °C and irreversible at higher temperatures. Thermoinactivation studies revealed that enzymatic activity diminished significantly at high temperatures, with greater loss of activity observed above 55 °C. Release of ammonia upon deamidation of YHB obeyed a similar temperature-dependence pattern. Dynamic light scattering and size exclusion-HPLC indicated formation of stable aggregates. Taking various findings on the influence of osmolytes and chaperone-like agents on YHB thermal denaturation together, it is proposed that the purely conformational transition of YHB is reversible, and irreversibility is due to aggregation, as a major cause. Deamidation of a critical Asn or Gln residue(s) may also play an important role. © 2008 Elsevier B.V. All rights reserved.
Thermally induced changes in the structure and activity of yeast hexokinase B
Rezaei-Ghaleh N.;
2008-01-01
Abstract
Yeast hexokinase has been poorly characterized in regard with its stability. In the present study, various spectroscopic techniques were employed to investigate thermal stability of the monomeric form of yeast hexokinase B (YHB). The enzyme underwent a conformational transition with a Tm of about 41.9 °C. The structural transition proved to be significantly reversible below 55 °C and irreversible at higher temperatures. Thermoinactivation studies revealed that enzymatic activity diminished significantly at high temperatures, with greater loss of activity observed above 55 °C. Release of ammonia upon deamidation of YHB obeyed a similar temperature-dependence pattern. Dynamic light scattering and size exclusion-HPLC indicated formation of stable aggregates. Taking various findings on the influence of osmolytes and chaperone-like agents on YHB thermal denaturation together, it is proposed that the purely conformational transition of YHB is reversible, and irreversibility is due to aggregation, as a major cause. Deamidation of a critical Asn or Gln residue(s) may also play an important role. © 2008 Elsevier B.V. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
