Structural requirements of alloxan and ninhydrin for glucokinase inhibition and of glucose for protection against inhibition

Abstract

1 In order to elucidate the mechanism underlying the interactions between glucose and alloxan when competing for the sugar binding site of glucokinase from pancreatic B-cells or liver, the structural requirements of the enzyme for inhibition by alloxan and for protection by glucose were determined. 2 With a half-maximal inhibitory concentration of 5 /M, alloxan was the most potent pyrimidine derivative inhibitor of glucokinase. Uramil was a less potent enzyme inhibitor. A variety of other pyrimidine derivatives and related substances were ineffective. 3 Ninhydrin also inhibited glucokinase with a half-maximal inhibitory concentration of 5 gM. Isatin was a slightly less potent enzyme inhibitor. Several other indoline derivatives were ineffective. 4 Only glucose derivatives with a sufficiently bulky substituent in position C-2, such as the glucokinase substrates glucose and mannose and the inhibitors mannoheptulose, glucosamine, and Nacetylglucosamine, protected glucokinase against inhibition by alloxan by binding to the active site of the enzyme. Glucose epimers which differed in other positions did not protect the enzyme against alloxan inhibition. 5 DTT (dithiothreitol) protected glucokinase against inhibition by alloxan and reversed the inhibition of the enzyme induced by alloxan. Thus the mechanism of glucokinase inhibition by alloxan and other inhibitors, such as uramil and ninhydrin, is an oxidation of functionally essential SH groups of the enzyme, where the most reactive keto group of the inhibitor acts as the hydrogen acceptor. The protective action of glucose and several C-2 epimers demonstrates that these functionally essential SH groups are situated in the sugar binding site of the glucokinase. 6 The present results support our contention, that the pancreatic B-cell glucokinase is the major target mediating the inhibition of insulin secretion by alloxan.

Alterations of insulin response to different β cell secretagogues and pancreatic vascular resistance induced by Nω‐nitro‐L‐arginine methyl ester

“…As concerns the mechanism by which NO causes inhibition, there is growing evidence that this short-lived oxygen free radical is able to react with a number of naturally occurring sulphydryl containing proteins (Stamler et al, 1992), and functionally essential SH groups in the glucose binding site of fi cell glucokinase have been shown to be a target for oxidising agents (Lenzen et al, 1988). In addition, NO also forms ironnitrosyl complexes with FeS containing enzymes such as aconitase, leading to reversible inactivation of the mitochondrial enzyme (Lancaster & Hibbs, 1990).…”

The mechanisms of alloxan- and streptozotocin-induced diabetes

“…Insulin biosynthesis is also inhibited by alloxan [47,48], most likely through the same mechanism. The insulin secretory response to other nutrient secretagogues, such as 2-ketoisocaproic acid and leucine, or non-nutrient secretagogues, such as the sulfonylurea drug tolbutamide, remains intact initially because it is not mediated via glucokinase [49], but is lost after a delay of up to 1 h [50] as a consequence of the gradual deterioration of beta cell function.…”

Alloxan: history and mechanism of action

“…Results from recent experiments indicate that glucokinase (also called hexokinase IV) is the enzyme in the pancreatic B cell and also in the liver, which is apparently most sensitive to inhibition by alloxan [41, 43,[45][46][47][48][49] and may be the primary target for ailoxan in the pancreatic B cell responsible for inhibition of glucoseinduced insulin secretion, and probably also for the pancreatic B cell toxic action of this agent [41]. The half-maximal inhibition of the enzyme is achieved by concentrations of alloxan well below 10 I.tmol/l for glucokinase from pancreatic B cells [43,49] as well as from liver [43,46,47,49], which are characterised by identical functional [43,49] and immunological [42,43] properties.…”