Site-directed mutagenesis studies on the determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase.
The determinants of sugar specificity and cooperative behavior of human beta-cell glucokinase were studied by mutating several active site residues and performing a steady-state kinetic analysis of the purified mutant and wild-type enzymes after their expression in Escherichia coli. Asn-204, Glu-256, and Glu-290 were predicted from molecular modeling to interact with the 3-OH, 4-OH, 2-OH, and 1-OH groups of glucose. Mutation of these residues resulted in enzymes with decreased values of kcat and increased values of Km for glucose, mannose, and 2-deoxyglucose. Lys-56 is also predicted to make an interaction with the side chain of Glu-256 and its mutation increased the Km for glucose, deoxyglucose, mannose, and fructose by 4-, 4-, 3-, and 10-fold, respectively, and also increased the kcat for fructose by 5-fold. The Ki values for N-acetylglucosamine and mannoheptulose for the wild-type enzyme were 0.2 and 0.8 mM, respectively, and mutation of glucose binding residues to alanine resulted in an increase of about 3 orders of magnitude in these Ki values. Mutation of residues that directly hydrogen bond glucose hydroxyls (Asn-204, Glu-256, and Glu-290) to alanine resulted in enzymes that did not exhibit cooperative behavior, but mutation of Lys-56 or other residues that do not directly contact glucose had no effect on the Hill coefficient. Only glucose and deoxyglucose exhibited cooperative behavior. The results 1) confirm the predictions of the model that Asn-204, Glu-256, and Glu-290 are important residues involved in catalysis and hydrogen bonding glucose hydroxyl groups, 2) provide evidence for a role of Lys-56 in hexose binding, and 3) are consistent with the cooperative behavior of glucokinase being mediated by interactions of other regions of the protein with the highly conserved active site glucose binding residues.
Department of Physiology and Biophysics, State University of New York, Stony Brook 11780.
L Z
LZ
Xu
W
W
Zhang
I T
IT
Weber
R W
RW
Harrison
S J
SJ
Pilkis
D000494:Allosteric Regulation; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000737:chemistry; D003847:Deoxyglucose / Q000378:metabolism; D005632:Fructose / Q000378:metabolism; D005941:Glucokinase / Q000737:chemistry / Q000378:metabolism*; D005944:Glucosamine; D006801:Humans; D007515:Islets of Langerhans / Q000201:enzymology; D008356:Mannoheptulose / Q000378:metabolism; D008358:Mannose / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D017931:DNA Primers; D005632:Fructose; D008356:Mannoheptulose; D003847:Deoxyglucose; D005941:Glucokinase; D005944:Glucosamine; D008358:Mannose