Skip to main content

Activation of the polyol pathway


Background

Sorbitol is formed from glucose NADPH + H+ + glucose → NADP+ + sorbitol catalyzed by aldose reductase (AR). This pathway is activated in hyperglycaemia because the KM of glucose for aldose reductase is high (70 mM). Sorbitol does not cross cell membranes, accumulates intracellularly and produces osmotic stress. Although marked sorbitol accumulation has been found in the lens of streptozotocin-induced diabetic rats, and aldose reductase inhibitors (ARIs) prevented this and cataract formation, similar high accumulation has not been found in tissues of diabetic human subjects and the therapeytic effects of AROs have, so far, been generally disappointing.

The activity of the polyol pathway may also contribute to the depletion of NAD+ in intracellular hyperglycaemia by transfer of reducing equivalents from the pentosephosphate pathway (by AR catalysed oxidation of NADPH) to the Embden-Meyerhof pathway (reduction of NAD+ to NADH catalysed by sorbitol dehydrogenase).

 polyol

Aldose Reductase (AR)

  • EC 1.1.1.21
  • Molecular mass 36 kDa, pI = 5.9
  • A broad specificity NADPH-dependent aldehyde reductase involved in aldehyde detoxification:
    • NADPH + H+ + RCHO → NADP+ + RCH2OH
  • AR reduces glucose to sorbitol, involved in osmoregulation under normal conditions and the development of diabetic complications in hyperglycaemia associated with diabetes mellitus
  • AR catalyses the dehydrogenation of 17-hydroxyprogesterone, found to be identical to 20,α-hydroxysteroid dehydrogenase (Biochemistry 32, 1401-1406, 1993)

Crystal Strcucture of Human AR

The crystal structure of human placental AR has been solved to 0.165 nm resolution (Science 257, 81-84, 1992).

 arcryst

Overview

Human AR belongs to a class of protein folds called TIM (Triosephosphate IsoMerase) barrel enzymes. The N-terminus contains S1 and S2 which form a hairpin β-sheet that covers the bottom of the barrel. S3 is the first of 8 parallel β-strands that form the interior of the barrel, connected by α-helices (H1-H8) (Biochemistry 33, 2011-2020,1994)

  • NADPH is bound in an extended conformation across the barrel with the nicotinamide ring centered in the deep part of the active site cavity and the adenosine-2'-monophosphate wedged in a shallow depression outside the barrel between a couple of β-strands and α-helices
  • The 4-pro-R hydrogen of the nicotinamide is transferred to the substrate

(Science 257, 81-84, 1992)

  • Important active site residues are: Tyr-48 (in the loop between S4 and H2), Lys-55 in S5, and His-110 in the loop between S6 and H4
  • Site specific mutagenesis suggests that Tyr-48 is the proton donor to the alcoholate anion; pKa of Tyr-48 is lower to 8.25
  • His-110 directs the stereochemistry of the hydride attack

(Biochemistry 33, 2011-2020 & 2012-2032,1994; ibid 34, 14374-14384, 1995)

Catalytic Mechanism

armech

(Biochemistry 33, 2011-2020,1994)

  • The aldehyde substrate binds with its carbonyl group in an active site pocket formed by His-110, Tyr-48 and the C4-N of the nicotinamide ring
  • This polarizes the carbonyl group for subsequent hydride attack and proton transfer from Tyr-48
  • Product release and cofactor exchange complete the cycle

Catalytic efficiency: re-appraisal of theories

  • AR binds both NADPH and NADP+ tightly: Ks = 0.05 mM and 0.08 mM, respectively. kcat/KM is diffusion-limited and varies little with aldehyde substrate - it is probably an idealized detoxification catalyst.
  • AR is primed for aldehyde reduction which is essentially a one-way, irreversible process because: kcat/KM is diffusion-limited, [NADPH]/[NADP+] » 50-500 under physiological conditions, and Ks for NADPH « physiological [NADPH]

The catalytic properties are primed for aldehyde reduction, and are well-suited for the physiological function (Biochemistry 31, 10139-10145, 1992)

The involvement of AR in diabetic complications and exploitation of this for a therapy has been investiagted in the development of aldose reductase inhibitors ARIs.

Return to: Diabetic Complications Hypotheses page

Next page: Summary of Strategies for Therapeutic Intervention


Return to the top of the page