The High Resolution Crystal Structure of Yeast Hexokinase PII with the Correct Primary Sequence Provides New Insights into Its Mechanism of Action

Abstract

Hexokinase 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. Two yeast hexokinase isozymes are known, namely PI and PII. The crystal structure of yeast hexokinase PII from Saccharomyces cerevisiae without substrate or competitive inhibitor is determined and refined in a tetragonal crystal form at 2.2-Å resolution. The folding of the peptide chain is very similar to that of Schistosoma mansoni and previous yeast hexokinase models despite only 30% sequence identity between them. Distinct differences in conformation are found that account for the absence of glucose in the binding site. Comparison of the current model with S. mansoni and yeast hexokinase PI structures both complexed with glucose shows in atomic detail the rigid body domain closure and specific loop movements as glucose binds. A hydrophobic channel formed by strictly conserved hydrophobic residues in the small domain of the hexokinase is identified. The channel's mouth is close to the active site and passes through the small domain to its surface. The possible role of the observed channel in proton transfer is discussed.

Crystal Structure of Hexokinase KlHxk1 of Kluyveromyces lactis

“…Domain Closure Motion, Ligand Binding, and CatalysisThe current knowledge of the domain closure motion of yeast hexokinases is based on and limited by the comparison of different isoenzymes of S. cerevisiae hexokinase; ScHxk1 is considered to represent the closed state (25,29), whereas ScHxk2 exhibits a comparatively open conformation (28,30). In contrast, the structural data presented in the present article were obtained with the single enzyme KlHxk1.…”

“…The interdomain rotation axis vector of the KlHxk1 domain movement between crystal forms IX and XI coincides closely (16°deviation, Fig. 4A) with the domain closure motion vector of the hexokinases of S. cerevisiae as inferred from a comparison of ScHxk2 (30) in the open conformation and ScHxk1 (29) in the closed conformation. This finding indicates that the three homologous hexokinases undergo similar domain movements during their catalytic cycles.…”

“…4A). It should be noted, however, that the latter two structures do not necessarily reflect a substrateinduced conformational transition because they belong to different isoenzymes of S. cerevisiae hexokinase (29,30), which share 77% sequence identity (22).…”

“…The latter situation may reflect either a disorder of the respective N-terminal residues or their proteolytic removal. Indeed, ScHxk2 crystal form BIII (PDB entry, 2YHX) (28) was grown from enzyme lacking the N-terminal 11 amino acids (61), whereas N-terminal integrity is unknown in the case of yeast hexokinase structures 1HKG (25), 3B8A (29), and 1IG8 (30).…”

“…More recently, structures have been determined to higher resolution for the two proteins. In the case of ScHxk1, the same crystal form was analyzed (PDB entry, 3B8A) (29), whereas a new crystal form was obtained for ScHxk2 (PDB entry, 1IG8) (30). Remarkably, in all of these structures the enzymes are present in the monomeric state, even though ScHxk1 and ScHxk2 are known to form dimers (31).…”