Thermally induced changes in the structure and activity of yeast hexokinase B.
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 T(m) of about 41.9 degrees C. The structural transition proved to be significantly reversible below 55 degrees 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 degrees 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.
Ramshini
Hassan
H
Institute of Biochemistry and Biophysics, University of Tehran, Tehran 13145-1384, Iran.
Rezaei-Ghaleh
Nasrollah
N
Ebrahim-Habibi
Azadeh
A
Saboury
Ali Akbar
AA
Nemat-Gorgani
Mohsen
M
D000577:Amides / Q000737:chemistry; D002364:Caseins / Q000737:chemistry; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004229:Dithiothreitol / Q000737:chemistry; D004795:Enzyme Stability; D006593:Hexokinase / Q000737:chemistry*; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D044366:Transition Temperature; D047408:gamma-Cyclodextrins / Q000737:chemistry
D000577:Amides; D002364:Caseins; D007527:Isoenzymes; D029701:Saccharomyces cerevisiae Proteins; D047408:gamma-Cyclodextrins; D006593:Hexokinase; D003545:Cysteine; C023792:gamma-cyclodextrin; D004229:Dithiothreitol