Aldose reductase, classified within the aldo-keto reductase family as AKR1B1, is an NADPH dependent enzyme that catalyzes the reduction of hydrophilic as well as hydrophobic aldehydes. AKR1B1 is the first enzyme of the so-called polyol pathway that allows the conversion of glucose into sorbitol, which in turn is oxidized to fructose by sorbitol dehydrogenase. The activation of the polyol pathway in hyperglycemic conditions is generally accepted as the event that is responsible for a series of long-term complications of diabetes such as retinopathy, cataract, nephropathy and neuropathy. The role of AKR1B1 in the onset of diabetic complications has made this enzyme the target for the development of molecules capable of inhibiting its activity. Virtually all synthesized compounds have so far failed as drugs for the treatment of diabetic complications. This failure may be partly due to the ability of AKR1B1 to reduce alkenals and alkanals, produced in oxidative stress conditions, thus acting as a detoxifying agent. In recent years we have proposed an alternative approach to the inhibition of AKR1B1, suggesting the possibility of a differential inhibition of the enzyme through molecules able to preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. The rationale and examples of this new generation of aldose reductase differential inhibitors (ARDIs) are presented.

A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme–substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (K_m=0.57 mM, k_cat=6.0×10³ min⁻¹ at pH 7.4 and 25 °C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed.

Aldose reductase (ALR2) is susceptible to oxidative inactivation by copper ion. The mechanism underlying the reversible modification of ALR2 was studied by mass spectrometry, circular dichroism, and molecular modeling approaches on the enzyme purified from bovine lens and on wild type and mutant recombinant forms of the human placental and rat lens ALR2. Two equivalents of copper ion were required to inactivate ALR2: one remained weakly bound to the oxidized protein whereas the other was strongly retained by the inactive enzyme. Cys³⁰³ appeared to be the essential residue for enzyme inactivation, because the human C303S mutant was the only enzyme form tested that was not inactivated by copper treatment. The final products of human and bovine ALR2 oxidation contained the intramolecular disulfide bond Cys²⁹⁸-Cys³⁰³. However, a Cys⁸⁰-Cys³⁰³ disulfide could also be formed. Evidence for an intramolecular rearrangement of the Cys⁸⁰-Cys³⁰³ disulfide to the more stable product Cys²⁹⁸-Cys³⁰³ is provided. Molecular modeling of the holoenzyme supports the observed copper sequestration as well as the generation of the Cys⁸⁰-Cys³⁰³disulfide. However, no evidence of conditions favoring the formation of the Cys²⁹⁸-Cys³⁰³ disulfide was observed. Our proposal is that the generation of the Cys²⁹⁸-Cys³⁰³ disulfide, either directly or by rearrangement of the Cys⁸⁰-Cys³⁰³ disulfide, may be induced by the release of the cofactor from ALR2 undergoing oxidation. The occurrence of a less interactive site for the cofactor would also provide the rationale for the lack of activity of the disulfide enzyme forms.