Thermally induced changes in the structure and activity of yeast hexokinase B Hassan Ramshini a , Nasrollah Rezaei-Ghaleh a , Azadeh Ebrahim-Habibi a , Ali Akbar Saboury a , Mohsen Nemat-Gorgani a,b, ⁎ a Institute of Biochemistry and Biophysics, University of Tehran, Tehran 13145-1384, Iran b Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA abstract article info Article history: Received 18 May 2008 Received in revised form 14 July 2008 Accepted 14 July 2008 Available online 19 July 2008 Keywords: Yeast hexokinase B Thermoinactivation Aggregation Deamidation Size exclusion-HPLC 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 °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. 1. Introduction Hexokinase (EC 2.7.1.1) is the first enzyme in the glycolytic pathway, catalyzing the transfer of a phosphoryl group from ATP to glucose to form glucose 6-phosphate and ADP. Hexokinases have been found in every organism checked, ranging from bacteria, yeast, and plants to humans and other vertebrates. They are categorized as actin fold proteins [1]. Four distinct mammalian isozymes designated as types I–IV have been characterized. While hexokinases types I and II bind to mitochondria through interaction with a porin known as voltage dependent anion channel (VDAC), type III and IV isozymes lack the hydrophobic N-terminal sequence which is required critically for enzyme binding to mitochondria [2]. The primary sequence alignment of a selection of proteins from the hexokinase family demonstrates extensive similarity between the N- and C-terminal halves of type I human hexokinase, rat hexokinase, and hexokinase from S. mansoni and between these and yeast hexokinase, consistent with the gene duplication-fusion concept proposed by Colowick [3]. There are two isoenzymes of hexokinase in yeast, A and B, with an overall homology in their amino acid sequences of about 76% [4]. They are structurally well characterized [4–7], showing a high degree of similarity in regard with tertiary structure [6,8,9]. Both of the isozymes share a similar α/β fold, and the polypeptide chain is distinctly folded into two domains of unequal size, the large and the small domain. These domains are separated by a large cleft forming the active site [10,11]. Each enzyme exists in monomer–dimer self- association equilibrium, with a dimer molecular weight of about 100 kDa. Dissociation is promoted by increases in pH, ionic strength, and temperature, and by a decrease in enzyme concentration [12,13]. The N-terminus of the protein is considered essential for its self- association [14]. Endogenous protease action during purification leads to the loss of 11 amino acids from the N-terminus, resulting in a predominantly monomeric form of about 50 kDa [15]. Yeast hexokinase B is the predominant hexose kinase in S. cerevisiae grown on glucose [16], involved in catabolite repression by glucose [17,18]. The enzyme exhibits regulatory properties at physiological pH values which include negative cooperativity with ATP, activation by citrate and some other anions [19,20]. Hexokinase malfunction has been implicated in a number of diseases in humans. For example, its activity has been reported to change significantly in patients with Alzheimer's disease [21,22] and markedly elevated in highly glycolytic, rapidly growing tumors [23,24]. Studies on protein stability is gaining more importance as the number of therapeutic protein products is increasing and protein stabilization is becoming more important due to their greater use under industrial conditions. As related to the present investigation, limited efforts have been directed toward elucidation of the mechan- isms associated with thermal stability of the mammalian [25] and yeast [26,27] hexokinases. In the present communication, we tried to elucidate the mechan- isms involved in thermal denaturation of yeast hexokinase B. Various Biophysical Chemistry 137 (2008) 88–94 ⁎ Corresponding author. Stanford Genome Technology Center, Stanford University, Palo Alto, CA, USA. Tel.: +1 650 812 1961; fax: +1 650 812 1975. E-mail address: mohsenn@stanford.edu (M. Nemat-Gorgani). 0301-4622/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bpc.2008.07.004 Contents lists available at ScienceDirect Biophysical Chemistry journal homepage: http://www.elsevier.com/locate/biophyschem