EFFECT OF MUTATIONS ON THE SENSITIVITY OF HUMAN BETA-CELL GLUCOKINASETO LIVER REGULATORY PROTEIN

Human beta-cell glucokinase and its liver counterpart displayed a half -saturating concentration of glucose (S-0.5) of about 8 mmol/l and a H ill coefficient of 1.7, and were as sensitive to inhibition by the rat liver regulatory protein as the rat liver enzyme. These results indic ate that the N-terminal region of glucokinase, which differs among the se three enzymes, is not implicated in the recognition of the regulato ry protein. They also suggest that the regulatory protein, or a relate d protein, could modulate the affinity of glucokinase for glucose in b eta cells. We have also tested the effect of several mutations, many o f which are implicated in maturity onset diabetes of the young, The mu tations affected the affinity for glucose and for the regulatory prote in to different degrees, indicating that the binding site for these mo lecules is different. An Asp(158)Ala mutation, found in the expression plasmid previously thought to encode the wild-type enzyme, increased the affinity for glucose by about 2.5-fold without changing the affini ty for the regulatory protein. The mutations that were found to decrea se the affinity for the regulatory protein (Asn(166)Arg, Val(203)Ala, Asn(204)Gln, Lys(414)Ala) clustered in the hinge region of glucokinase and nearby in the large and small domains. These results are in agree ment with the concept that part of the binding site for the regulatory protein is situated in the hinge region of this enzyme.