Human Aldose Reductase: Subtle Effects Revealed by Rapid Kinetic Studies of the C298A Mutant Enzyme

Grimshaw, Charles E.; Bohren, Kurt M.; Lai, Chung-Jeng; Gabbay, Kenneth H.

doi:10.1021/bi00044a013

Cited by 39 publications

(33 citation statements)

References 30 publications

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“…The C298A mutant of human aldose reductase has a high enzyme activity. V͞E t of C298A is 8.7 times that of wild type (9). Nevertheless, the effects of urea and the methylamines on enzyme activity are qualitatively similar, comparing the wild type and mutant.…”

Section: Both Urea and Methylamines Inhibit Human Recombinantmentioning

confidence: 85%

“…The K m of the C298A mutant is considerably higher than that of the wild type (9). In the present studies the mean values with DL-glyceraldehyde as substrate are wild type, 0.157 Ϯ 0.005 mM, and C298A, 1.03 Ϯ 0.06 mM, and with D-xylose as substrate the values are wild type, 8.1 Ϯ 0.3 mM, and C298A, 218.3 Ϯ 7.6 mM.…”

Section: Both Urea and Methylamines Inhibit Human Recombinantmentioning

confidence: 86%

“…Mutation of a single amino acid (C298A) in recombinant human aldose reductase increases the K m for D-xylose by 37-fold and also increases V max (9). The increase in K m is mainly caused by increase in the rate constant for *E⅐NADP ϩ 3 E⅐NADP ϩ , resulting in relaxation of a tight binary E⅐nucleotide complex to a more weakly bound complex.…”

Section: Fig 5 Effect Of Urea and Gpc Onmentioning

confidence: 99%

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Factors affecting counteraction by methylamines of urea effects on aldose reductase

Burg

Peters

Bohren

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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The concentration of urea in renal medullary cells is high enough to affect enzymes seriously by reducing V max or raising K m , yet the cells survive and function. The usual explanation is that the methylamines found in the renal medulla, namely glycerophosphocholine and betaine, have actions opposite to those of urea and thus counteract its effects. However, urea and methylamines have the similar (not counteracting) effects of reducing both the K m and V max of aldose reductase (EC 1.1.1.21), an enzyme whose function is important in renal medullas. Therefore, we examined factors that might determine whether counteraction occurs, namely different combinations of assay conditions (pH and salt concentration), methylamines (glycerophosphocholine, betaine, and trimethylamine N-oxide), substrates (DLglyceraldehyde and D-xylose), and a mutation in recombinant aldose reductase protein (C298A). We find that V max of both wild-type and C298A mutant generally is reduced by urea and͞or the methylamines. However, the effects on K m are much more complex, varying widely with the combination of conditions. At one extreme, we find a reduction of K m of wild-type enzyme by urea and͞or methylamines that is partially additive, whereas at the other extreme we find that urea raises K m for D-xylose of the C298A mutant, betaine lowers the K m , and the two counteract in a classical fashion so that at a 2:1 molar ratio of betaine to urea there is no net effect. We conclude that counteraction of urea effects on enzymes by methylamines can depend on ion concentration, pH, the specific methylamine and substrate, and identity of even a single amino acid in the enzyme.High concentrations of urea are present in the tissues of marine elasmobranchs and in the mammalian renal medulla. Urea generally destabilizes biological macromolecules, altering their structure and function. Such effects are expected to be deleterious. However, the urea-rich tissues also contain high concentrations of certain methylamine compounds, principally trimethylamine N-oxide (TMAO) in elasmobranchs (1) and glycine betaine (betaine) and glycerophosphocholine (GPC) in mammalian renal medulla (2). These methylamines are believed to protect the cells from urea by stabilizing macromolecules and thus counteracting the actions of urea. The two effects are independently additive (1, 3). When the ratio of methylamines to urea is appropriate (often 1:2), their opposing effects are reported to counteract, preserving macromolecular structure and function. The theory of counteracting osmolytes is strongly supported by the occurrence of methylamines in organisms and tissues containing high concentrations of urea (4), by survival in tissue culture of cells containing high levels of both urea and betaine, compared with the poor survival of cells containing only one or the other (5), and by many observations on isolated macromolecules in vitro (4).However, we recently found that three different methylamines, namely TMAO, betaine, and GPC, do not counteract inhibition by ure...

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Section: Both Urea and Methylamines Inhibit Human Recombinantmentioning

confidence: 85%

Section: Both Urea and Methylamines Inhibit Human Recombinantmentioning

confidence: 86%

Section: Fig 5 Effect Of Urea and Gpc Onmentioning

confidence: 99%

See 1 more Smart Citation

Factors affecting counteraction by methylamines of urea effects on aldose reductase

Burg

Peters

Bohren

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…The polyol pathway enzyme aldose reductase catalyzes the reduction of aldo sugars and other saturated and unsaturated aldehydes [16][17][18][19][20]. This enzyme constitutes the first and the rate limiting step of the polyol pathway.…”

Section: Introductionmentioning

confidence: 99%

Polyol pathway and modulation of ischemia-reperfusion injury in Type 2 diabetic BBZ rat hearts

Hwang

Ananthakrishnan

et al. 2008

Cardiovasc Diabetol

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We investigated the role of polyol pathway enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH) in mediating injury due to ischemia-reperfusion (IR) in Type 2 diabetic BBZ rat hearts. Specifically, we investigated, (a) changes in glucose flux via cardiac AR and SDH as a function of diabetes duration, (b) ischemic injury and function after IR, (c) the effect of inhibition of AR or SDH on ischemic injury and function. Hearts isolated from BBZ rats, after 12 weeks or 48 weeks diabetes duration, and their non-diabetic littermates, were subjected to IR protocol. Myocardial function, substrate flux via AR and SDH, and tissue lactate:pyruvate (L/P) ratio (a measure of cytosolic NADH/NAD + ), and lactate dehydrogenase (LDH) release (a marker of IR injury) were measured. Zopolrestat, and CP-470,711 were used to inhibit AR and SDH, respectively. Myocardial sorbitol and fructose content, and associated changes in L/P ratios were significantly higher in BBZ rats compared to non-diabetics, and increased with disease duration. Induction of IR resulted in increased ischemic injury, reduced ATP levels, increases in L/P ratio, and poor cardiac function in BBZ rat hearts, while inhibition of AR or SDH attenuated these changes and protected hearts from IR injury. These data indicate that AR and SDH are key modulators of myocardial IR injury in BBZ rat hearts and that inhibition of polyol pathway could in principle be used as a therapeutic adjunct for protection of ischemic myocardium in Type 2 diabetic patients.

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“…In aldose reductase, Cys-298 stabilizes the closed conformation of a nucleotide-enfolding loop that locks the coenzyme into place (Borhani et al 1992;Grimshaw et al 1995a). The sulfhydryl moiety of Cys-298 is directed into the catalytic center of the protein, where it is in close contact with the C4 position of the nucleotide cofactor (Wilson et al 1992;Bohren et al 1994;Harrison et al 1994).…”

Section: Structurally and Functionally Important Residues In B-crystamentioning

confidence: 99%

Evolution of the Aldose Reductase-Related Gecko Eye Lens Protein ρB-Crystallin: A Sheep in Wolf's Clothing

et al. 2001

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Abstract. B-crystallin (AJ245805) is a major protein component (20%) in the eye lens of the gecko Lepidodactylus lugubris. Limited peptide sequence analysis earlier revealed that it belongs to the aldo-keto reductase superfamily, as does the frog lens -crystallin. We have now determined the complete cDNA sequence of Bcrystallin and established that it is more closely related to the aldose reductase branch of the superfamily than to frog -crystallin. These gecko and frog lens proteins have thus independently been recruited from the same enzyme superfamily. Aldose reductase is implicated in the development of diabetic cataract in mammals, and, if active, B-crystallin might be a potential risk for the gecko lens. Apart from a replacement 298 Cys → Tyr, B-crystallin possesses all amino acid residues thought to be required for catalytic activity of the aldose reductases. However, modeling studies of the B-crystallin structure indicate that substrate specificity and nicotinamide cofactor affinity might be affected. Indeed, neither recombinant B-crystallin nor the reverse mutant 298 Tyr → Cys showed noticeable activity toward aliphatic and aromatic substrates, although cofactor binding was retained. Various other oxidoreductases are known to be recruited as abundant lens proteins in many vertebrate species; B-crystallin demonstrates that an aldose reductase-related enzyme also can be modified to this end.

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Human Aldose Reductase: Subtle Effects Revealed by Rapid Kinetic Studies of the C298A Mutant Enzyme

Cited by 39 publications

References 30 publications

Factors affecting counteraction by methylamines of urea effects on aldose reductase

Factors affecting counteraction by methylamines of urea effects on aldose reductase

Polyol pathway and modulation of ischemia-reperfusion injury in Type 2 diabetic BBZ rat hearts

Evolution of the Aldose Reductase-Related Gecko Eye Lens Protein ρB-Crystallin: A Sheep in Wolf's Clothing

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