Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose.

Abstract

To identify the amino acids involved in the specific regulatory properties of glucokinase, and particularly its low affinity for glucose, mutants of the human islet enzyme have been prepared, in which glucokinase-specific residues have been replaced. Two mutations increased the affinity for glucose by twofold (K296M) and sixfold (Y214A), the latter also decreasing the Hill c o e fficient from 1.75 to 1.2 with minimal change in the a ffinity for AT P. Combining these two mutations with N166R resulted in a 50-fold decrease in the half-saturating substrate concentration (S 0 . 5 ) value, which became then comparable to the K m of hexokinase II. The location of N166, Y214, and K296 in the three-dimensional structure of glucokinase suggests that these mutations act by favoring closure of the catalytic cleft. As a rule, mutations changed the affinity for glucose and for the competitive inhibitor mannoheptulose (MH) in parallel, whereas they barely affected the affinity for N-acetylglucosamine (NAG). These and other results suggest that NAG and MH bind to the same site but to d i fferent conformations of glucokinase. A small reduction in the affinity for the regulatory protein was observed with mutations of residues on the smaller domain and in the hinge region, confirming the bipartite nature of the binding site for the regulatory protein. The K296M mutant was found to have a threefold decreased affinity for palmitoyl CoA; this effect was additive to that previously observed for the E279Q mutant, indicating that the binding site for long-chain acyl CoAs is located on the upper face of the larger domain. D i a b e t e s 4 9 :1 9 5-201, 2000

Stimulation of Hepatocyte Glucose Metabolism by Novel Small Molecule Glucokinase Activators

“…Moreover, we demonstrate the efficacy of these compounds in stimulating glucose phosphorylation, glycolysis, and glycogen synthesis in hepatocytes. Furthermore, we show that the compounds affect the affinity for mannoheptulose but not for three other GK inhibitors, including GKRP, which suggests a similar mechanism of action of the compounds to activating mutations of the GK gene (8,9,32). The compounds cause translocation of GK from the nucleus despite the fact that they do not dissociate GK from GKRP.…”

“…GKA1 did not affect the affinity for N-acetylglucosamine, which binds to the catalytic site (35), or for palmitoyl-CoA and GKRP, which bind to allosteric sites (6). However, it increased the affinity for mannoheptulose, which is thought to bind to the catalytic site (31,32). This glucose analog differs from N-acetylglucosamine in its effects on the cooperativity of GK for glucose (36).…”

“…This glucose analog differs from N-acetylglucosamine in its effects on the cooperativity of GK for glucose (36). This has been explained by binding of these inhibitors to the open (N-acetylglucosamine) or closed (mannoheptulose) conformations of GK (31,32). Further insight into the mechanism of action of the GK activators can be envisaged from recent studies on the kinetic properties of GK mutants.…”

“…However, mutations of residues distant from the catalytic site affect the affinity for glucose and mannoheptulose but not N-acetylglucosamine (31,37). Most, though not all, of the activating mutations are in the hinge region of the bipartite structure (8,9,32,38). It is assumed that they favor the closed conformation, which has a higher affinity for glucose.…”

Insights Into the Biochemical and Genetic Basis of Glucokinase Activation From Naturally Occurring Hypoglycemia Mutations

“…This is seen in the father of family 2, who is asymptomatic despite having a lifetime of untreated fasting glucose values between 2.7 and 3.1 mmol/l. Activating mutations with kinetic changes that result in higher relative activities than those described in patients to date can be predicted from the artificial mutations with more dramatic increases in relative activity indexes (23). Such severe mutant enzymes, although still regulated by glucose, may result in more pronounced hypoglycemia, and patients might not respond sufficiently to diazoxide and have severe neuroglycopenic symptoms.…”