From Clinicogenetic Studies of Maturity-Onset Diabetes of the Young to Unraveling Complex Mechanisms of Glucokinase Regulation

Abstract

Glucokinase functions as a glucose sensor in pancreatic ␤-cells and regulates hepatic glucose metabolism. A total of 83 probands were referred for a diagnostic screening of mutations in the glucokinase (GCK) gene. We found 11 different mutations (V62A, G72R, L146R, A208T, M210K, Y215X, S263P, E339G, R377C, S453L, and IVS5 ؉ 1G>C) in 14 probands. Functional characterization of recombinant glutathionyl S-transferase-G72R glucokinase showed slightly increased activity, whereas S263P and G264S had near-normal activity. The other point mutations were inactivating. S263P showed marked thermal instability, whereas the stability of G72R and G264S differed only slightly from that of wild type. G72R and M210K did not respond to an allosteric glucokinase activator (GKA) or the hepatic glucokinase regulatory protein (GKRP). Mutation analysis of the role of glycine at position 72 by substituting E, F, K, M, S, or Q showed that G is unique since all these mutants had very low or no activity and were refractory to GKRP and GKA. Structural analysis provided plausible explanations for the drug resistance of G72R and M210K. Our study provides further evidence that protein instability in combination with loss of control by a putative endogenous activator and GKRP could be involved in the development of hyperglycemia in maturity-onset diabetes of the young, type 2. Furthermore, based on data obtained on G264S, we propose that other and still unknown mechanisms participate in the regulation of glucokinase.

Functional analysis of human glucokinase gene mutations causing MODY2: exploring the regulatory mechanisms of glucokinase activity

“…Our functional study indicates that the R397L mutation has almost no effects on GCK kinetics. Although we found some effect of this mutation on the thermal stability of the enzyme, these effects were relatively mild and not as pronounced as those induced by mutation R308W or previously described mutations such as E265K, E300K or S263P [17,19,31]. It is possible that this mild thermal lability alone might not be sufficient to account for the hyperglycaemia observed in the patients.…”

“…It is possible that this mild thermal lability alone might not be sufficient to account for the hyperglycaemia observed in the patients. Loss of regulation by GCKR and allosteric activators has been reported for other GCK mutants with increased or nearly-normal enzyme activity [18,31]. Despite this, we found that the R397L mutation did not affect GCK-GCKR binding in vitro and was not located in the allosteric site predicted from the crystal structure [30].…”

“…Interestingly, the crystal structure of GCK complexed with a GCK allosteric activator indicates that the M235 residue, together with residues V62, I159, M210, I211 and V452, is involved in hydrophobic interactions with this compound [30]. As expected from this model, MODY2 mutations V62M and M210K result in a loss of effect of GCK activators as well as a loss of inhibition by GCKR [18,31]. It has been proposed that these effects could be responsible for the development of hyperglycaemia.…”

“…The binding of GCKR to GCK is responsible for the inhibition and translocation of the enzyme into the nucleus. Although the physiological importance of GCK regulation by GCKR has been known for sometime, only a few MODY2 mutations have been reported to affect this regulation [18,28,31]. However, and contrary to what might be expected a priori, most of those mutations reduced GCK inhibition by the regulatory protein.…”

The previously reported T342P GCK missense variant is not a pathogenic mutation causing MODY

“…In some instances these mutations have been shown to alter enzyme regulation by small molecular activators or glucokinase regulatory protein. In these cases, however, there has been clear support for pathogenicity from co-segregation studies [5,8]. For the remaining variants where co-segregation has not been provided it is possible that these variants, like T342P, are rare polymorphisms.…”