Active site residues of human brain hexokinase as studied by site-specific mutagenesis.
The truncated gene of hexokinase, mini-hexokinase, starting with methionine 455 and ending at the C terminus was expressed in Escherichia coli. Mini-hexokinase lost its ability to ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the presence of phosphate (P(i)). We suggest that the P(i) site either resides in the N-terminal half of hexokinase I or requires the N-terminal portion of the enzyme. Site-directed mutagenesis was performed to obtain two mutants of mini-hexokinase: C606S and C628S. Both are thought to be associated with the active site of hexokinase I. These mutants exhibited a 3-fold increase in Km for glucose but no change in either the Km for ATP or the kcat. The circular dichroism (CD) spectra showed no differences among the wild-type or mutant enzymes. These results suggest that Cys606 and Cys628 are not involved in glucose binding directly. The putative ATP-binding site of full-length human brain hexokinase may involve Arg539 and Gly679, and these residues were mutated to Ile. For the mutant R539I, the kcat value decreased 114-fold relative to wild-type hexokinase, whereas the Km values for ATP and glucose changed only slightly. No change was observed in the Ki value for 1,5-anhydroglucitol 6-phosphate. CD spectra showed only a slight change in secondary structure. For the mutant G679I, overexpressed hexokinase is insoluble. We suggest that Arg539 is important for catalysis because it stabilizes the transition state product ADP-hexokinase. Gly679 is probably important for proper folding of the protein.
Zeng
C
C
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011, USA.
Fromm
H J
HJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D005947:Glucose / Q000378:metabolism; D005998:Glycine / Q000737:chemistry; D006593:Hexokinase / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D013329:Structure-Activity Relationship
D017931:DNA Primers; D000255:Adenosine Triphosphate; D001120:Arginine; D006593:Hexokinase; D005947:Glucose; D003545:Cysteine; D005998:Glycine