ACTIVE-SITE RESIDUES OF HUMAN BRAIN HEXOKINASE AS STUDIED BY SITE-SPECIFIC MUTAGENESIS

作者：ZENG CB; FROMM HJ

来源：Journal of Biological Chemistry, 1995, 270(18): 10509-10513.

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 K-m for glucose but no change in either the K-m for ATP or the k(cat). The circular dichroism (CD) spectra showed no differences among the wild-type or mutant enzymes. These results suggest that Cys(606) and Cys(628) are not involved in glucose binding directly. The putative ATP-binding site of full-length human brain hexokinase may involve Arg(539) and Gly(679), and these residues were mutated to Ile. For the mutant R539I, the k(cat) value decreased 114-fold relative to wildtype hexokinase, whereas the K-m values for ATP and glucose changed only slightly. No change was observed in the K-i 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 Arg(539) is important for catalysis because it stablizes the transition state product ADP-hexokinase. Gly(679) is probably important for proper folding of the protein.

• 出版日期1995-5-5