Spectroscopic and kinetic characterization of nonenzymic and aldose reductase mediated covalent NADP-glycolaldehyde adduct formation

Abstract

Reaction of glycolaldehyde with the binary E-NADP complex of bovine kidney aldose reductase (ALR2) produces an enzyme-bound chromophore whose absorbance (lambd max 341 nm) and fluorescence (lambda ex max 341 nm; lambda emit max 421 nm) properties are distinct from those of NADPH or E.NADPH yet are consistent with the proposed covalent adduct structure [1,4-dihydro-4-(1-hydroxy-2-oxoethyl)nicotinamide adenine dinucleotide phosphate]. The kinetics of adduct formation, both in solution and at the enzyme active site, support a mechanism involving rate-determining enolization of glycolaldehyde at high [NADP+] or [E.NADP]. At low [NADP+] or [E.NADP] the reaction is second-order overall, but the ALR2-mediated reaction displays saturation by glycolaldehyde due to competition of the aldehyde (plus hydrate) and enol for E.NADP. Measurement of the pre-steady-state burst of E-adduct formation confirms that glycolaldehyde enol is the reactive species and gives a value of 1.3 x 10(-6) for Kenol = [enol]/[( aldehyde] + [hydrate]), similar to that determined by trapping the enol with I3-. At the ALR2 active site, the rate of adduct formation is enhanced 79,000-fold and the adduct is stabilized greater than or equal to 13,000-fold relative to the reaction with NADP+ in solution. A portion of this enhancement is ascribed to specific interaction of NADP+ with the enzyme since the 3-acetylpyridine analogue, (AP)ADP+, gives values that are 15-200-fold lower. Additional evidence for strong interaction of ALR2 with both NADP+ and NADPH is reported. Yet, because dissociation of adduct is slow, catalysis of the overall adduct formation reaction by ALR2 is less than or equal to 67-fold.

Mechanistic studies of morphine dehydrogenase and stabilization against covalent inactivation

“…A sequential ordered mechanism with cofactor binding first has been demonstrated for aldose reductase [25,26], aldehyde reductase [27] and 3α-hydroxysteroid dehydrogenase [23], although, in the cases of aldose reductase and hydroxysteroid dehydrogenase, alternative evidence has suggested a random binding mechanism and\or formation of enzyme-substrate complexes in the absence of cofactor under some circumstances. In the case of MDH, inhibition by codeine of codeinone reduction at pH 7 is competitive with respect to codeinone, suggesting that codeinone is able to prevent codeine from binding to the enzyme-NADP + complex.…”

“…An alternative possibility is that the reaction displays Theorell-Chance kinetics, with reaction and alkaloid product dissociation occurring much more rapidly than cofactor release. This is plausible, since cofactor release in aldose reductase has been shown to require an isomerization of the enzyme-NADP + complex [25,26] which is rate-limiting in the direction of aldose reduction.…”

Aldose reductase: model for a new paradigm of enzymic perfection in detoxification catalysts

“…Given a kcat of 0.2 s™1, this means an effective capacity of 0.5 µ /s for reduction of glyceraldehyde present at 20 µ . Such a capacity is apparently sufficient to ensure the removal of spurious aldehydes that might otherwise react with free amino groups on various proteins (Acharya et al, 1988), on NAD-(P) nucleotides (Grimshaw et al, 1990a), or on the adenine and guanine bases in DNA (McGhee & von Hippel, 1975).…”

The atomic resolution structure of human aldose reductase reveals that rearrangement of a bound ligand allows the opening of the safety-belt loop

“…The mechanism for the forward reaction catalyzed by AR can be described as a sequential ordered mechanism (Grimshaw et al, 1990a(Grimshaw et al, , 1995Kubiseski et al, 1992) in which NADPH binds first, followed by the binding of the aldehyde substrate. After conversion of the aldehyde into an alcohol, the product of the reaction is released and the oxidized cofactor disengages from the enzyme.…”

“…In the forward reaction, an initial isomerization takes place after the binding of the cofactor (Kubiseski et al, 1992), while a second isomerization occurs after the reaction is completed but prior to the release of the cofactor (Grimshaw et al, 1990b). Furthermore, kinetic experiments have shown that both the reduced and oxidized version of the cofactor bind very tightly to the enzyme, with K d values smaller than 100 nM (Grimshaw et al, 1990a;Kubiseski et al, 1992), and that the conformational change preceding the dissociation of the cofactor is the overall rate-limiting step in the enzymatic cycle (Grimshaw et al, 1995).…”