| Many forms of diabetes can be treated, however, complication from diabetes remains one of the major causes of death by disease in the United States today. Among the potentially serious complications are blindness and cardiovascular disease. These symptoms may occur as a consequence of some abnormalities in carbohydrate metabolism. In particular the polyol metabolic pathway and the levels of the metabolic intermediate sorbitol have been implicated. The structure and specificity of the two enzymes that catalyze the metabolic interconversions in this pathway, aldose reductase and sorbitol dehydrogenase, have been examined. Through the use of alternative structures, specifically fluorosugars and deoxysugars, the active site of these enzymes were probed by using both classical kinetic studies and protein molecular modeling studies. In the case of aldose reductase, all of the deoxy and fluorodeoxy compounds that were examined were found to be substrates. The 3- and 4-fluoro analogues were ten to fifty-fold better substrates than the physiological substrate glucose. This overall enhancement is most likely due to a dipole effect of fluorine when this compound binds in the active site. Although this result was unexpected in the series of compounds that were studied, the observation is consistent with the broad specificity of aldose reductase, ranging from aliphatic to aromatic aldehydes. The substrate pattern for the sorbitol analogues with sorbitol dehydrogenase shows decreased binding for the 3- and 4-fluoro analogues, but enhanced binding for both the 6-deoxy and 6-fluoro substrate. The surprising result is that the 6-deoxy compound, which can neither donate nor accept a hydrogen bond, is the best substrate in this series of compounds. This 6-position is far removed from the site of reactivity, and is not thought to be involved in the reaction, since the five carbon analogue xylitol is a very good substrate. Molecular modeling studies suggest a more hydrophobic area around this portion of sorbitol binding site, which may better accommodate the binding of the deoxy substrate. The results of these studies serve to ascertain some of the details of how each of these enzymes in the polyol pathway function. |
