Biomedical subjects

D K Wilson

Publications and source records attributed to D K Wilson.

At least 37 records · Page 2Linked to original sources

PubMed1996

Specifically targeted modification of human aldose reductase by physiological disulfides.

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

Aldehyde Reductase↗

PubMed1996

Site-directed mutagenesis of histidine 238 in mouse adenosine deaminase: substitution of histidine 238 does not impede hydroxylate formation.

His 238, a conserved amino acid located in hydrogen-bonding distance from C-6 of the substrate in the active site of murine adenosine deaminase (mADA) and postulated to play an important role in catalysis, was altered into an alanine, a glutamate, and an arginine using site-directed mutagenesis. The Ala and Glu substitutions did not result in changes of the secondary or tertiary structure, while the Arg mutation caused local perturbations in tertiary structure and quenched the emission of one or more enzyme tryptophans. Neither the Glu or Arg mutations affected substrate binding affinity. By contrast, the Ala mutation enhanced substrate and inhibitor binding by 20-fold. The most inactive of the mutants, Glu 238, had a kcat/K(m) 4 x 10(-6) lower than the wild-type value, suggesting that a positive charge on His 238 is important for proper catalytic function. The Ala 238 mutant was the most active ADA, with a kcat/K(m) 2 x 10(-3) lower than the wild-type value. NMR spectroscopy and crystallography revealed that this mutant is able to catalyze hydration of purine riboside, a ground-state analog of the reaction. These results collectively show that His 238 is not required for formation of the hydroxylate used in the deamination and may instead have an important electrostatic role.

Adenosine Deaminase↗

PubMed1996

Probing the functional role of two conserved active site aspartates in mouse adenosine deaminase.

Two adjacent aspartates, Asp 295 and Asp 296, playing major roles in the reaction catalyzed by mouse adenosine deaminase (mADA) were altered using site-directed mutagenesis. These mutants were expressed and purified from an ADA-deficient bacterial strain and characterized. Circular dichroism spectroscopy shows the mutants to have unperturbed secondary structure. Their zinc content compares well to that of wild-type enzyme. Changing Asp 295 to a glutamate decreases the kcat but does not alter the Km for adenosine, confirming the importance of this residue in the catalytic process and its minimal role in substrate binding. The crystal structure of the D295E mutant reveals a displacement of the catalytic water from the active site due to the longer glutamate side chain, resulting in the mutant's inability to turn over the substrate. In contrast, Asp 296 mutants exhibit markedly increased Km values, establishing this residue's critical role in substrate binding. The Asp 296->Ala mutation causes a 70-fold increase in the Km for adenosine and retains 0.001% of the wild-type kcat/Km value, whereas the ASP 296->Asn mutant has a 10-fold higher Km and retains 1% of the wild-type kcat/Km value. The structure of the D296A mutant shows that the impaired binding of substrate is caused by the loss of a single hydrogen bond between a carboxylate oxygen and N7 of the purine ring. These results and others discussed below are in agreement with the postulated role of the adjacent aspartates in the catalytic mechanism for mADA.

Adenosine Deaminase↗

PubMed1996

Theoretical study of inhibition of adenosine deaminase by (8R)-coformycin and (8R)-deoxycoformycin.

Molecular dynamics and free energy simulations were performed to examine the binding of (8R)-deoxycoformycin and (8R)-coformycin to adenosine deaminase. The two inhibitors differ only at the 2' position of the sugar ring; the sugar moiety of conformycin is ribose, while it is deoxyribose for deoxycoformycin. The 100 ps molecular dynamics trajectories reveal that Asp 19 and His 17 interact strongly with the 5' hydroxyl group of the sugar moiety of both inhibitors and appear to play an important role in binding the sugar. The 2' and 3' groups of the sugars are near the protein-water interface and can be stabilized by either protein residues or water. The flexibility of the residues at the opening of the active site helps to explain the modest difference in binding of the two inhibitors and how substrates/inhibitors can enter an otherwise inaccessible binding site.

Adenosine Deaminase↗

PubMed1996

Variability in salt sensitivity classifications in black male versus female adolescents.

Salt sensitivity (changes in blood pressure in response to alterations in salt intake) may be a risk factor for hypertension. In the present study, we examined the prevalence of salt sensitivity based on two different classifications in healthy black male and female adolescents (aged 13 to 16 years). A total of 135 black adolescents participated in a 50 mmol/24 h low sodium diet for 5 days and a 150 mmol/24 h NaCl supplement for 10 days. Dietary compliance was defined as sodium excretion less than or equal to 50 mmol/24 h for the low sodium diet and greater than or equal to 165 mmol/24h for the high NaCl supplement. Salt sensitivity was defined by two classifications: (1) as a decrease in mean blood pressure greater than or equal to 5 mm Hg from baseline to the low sodium diet, and (2) as an increase in mean blood pressure greater than or equal to 5 mm Hg from the low sodium diet to the high NaCl supplement. With classification 1, 14% of boys were identified as salt sensitive compared with 22% of girls. With classification 2, however, 31% of boys were identified as salt sensitive compared with 18% of girls. Analyses based on changes in systolic pressure demonstrated similar findings across sex, although overall classifications based on systolic pressure yielded a greater percentage of salt-sensitive subjects. These sex differences in classification patterns were not due to differences in other important variables, such as changes in sodium excretion, potassium excretion, or Quetelet index. These results suggest that the prevalence of salt sensitivity differs by sex depending on the type of protocol used for the classification of salt sensitivity in a black pediatric population.

Adolescent↗

PubMed1995

1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldo-keto reductase family, complexed with coenzyme and inhibitor.

Murine FR-1 is a protein that is induced by fibroblast growth factor-1 and, therefore, may play a role in the regulation of the cell cycle. Sequence comparison indicates that it is a member of the NADPH-dependent aldo-keto reductase family. It bears 70% identity to human aldose reductase, an enzyme implicated in diabetic complications and a target for drug design. We have determined the 1.7 A resolution structure of the FR-1 in a ternary complex with NADPH and zopolrestat, a potent aldose reductase inhibitor. FR-1 folds into a (beta/alpha)8 barrel with an active site characterized by a preponderance of hydrophobic residues residing in a deep oblong cavity at the C-terminal end of the beta-barrel. The nicotinamide moiety of the coenzyme sits in the base of the cavity. Zopolrestat occupies the active site cavity and makes numerous contacts with several hydrophobic residues. The FR-1 ternary complex structure indicates that it uses the same general catalytic mechanism as aldose reductase and other members of the family whose structures have been determined. The protein exhibits reductase activity with DL-glyceraldehyde as a substrate and is strongly inhibited by zopolrestat. When compared with the structure of a similar ternary complex of aldose reductase, the binding site retains many of the interactions with the coenzyme and inhibitor from the conserved residues. Some differences in sequence, however, create a larger binding site that contains six more water molecules than in the aldose reductase ternary complex structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Oxidoreductases↗

PubMed1994

Crystallographic observation of a trapped tetrahedral intermediate in a metalloenzyme.

Formation of tetrahedral transition intermediates is a key step in many enzyme catalyzed reactions. Much of our understanding of these and other intermediates, at the atomic level, has come from crystallographic studies of very few enzymes with bound, synthetic, transition-state analogues. Here we present the structure of adenosine deaminase, a zinc-metalloenzyme critical in both purine metabolism and development of the lymphoid system, having performed a stereospecific hydroxide addition to the C6 of inosine. This addition causes the O6 oxygen of inosine to assume an orientation analogous to the position of the amino leaving group of the tetrahedral intermediate in the enzyme-catalyzed hydrolytic deamination of adenosine to inosine.

Adenosine Deaminase↗

PubMed1994

Race and sex differences in health locus of control beliefs and cardiovascular reactivity.

Examined the effects of race, sex, health locus of control (HLC), and the interactions of these variables on cardiovascular reactivity in 214 children. Participants completed the Multidimensional HLC scales for children. Systolic and diastolic blood pressures were measured at rest and during a reactivity task. Black boys had significantly higher scores on the powerful others (external HLC) subscale (p < 0.01) than white boys and girls. Furthermore, black boys showed greater systolic blood pressure reactivity when they were low on internality (vs. high) as compared to the other race-sex groups. Social and cultural factors may explain these differential findings across race and sex.

Adolescent↗

PubMed1994

Aldose reductase catalysis and crystallography. Insights from recent advances in enzyme structure and function.

Enhanced metabolism of glucose via the polyol pathway may play an important role in the pathogenesis of diabetic retinopathy, neuropathy, and nephropathy. Aldose reductase catalyzes the NADPH-dependent conversion of glucose to sorbitol, the first step in the polyol pathway. Interruption of the polyol pathway by inhibition of aldose reductase holds considerable promise as a therapeutic measure to prevent or delay the onset and severity of these late complications of diabetes. Dramatic advances in our understanding of the molecular biology, enzymology, and three-dimensional structure of aldose reductase have occurred in recent years, providing new and challenging insights into the enzyme's catalytic mechanism. Recent developments in structure determination of aldose reductase and the implications for evaluation and development of aldose reductase inhibitors are summarized.

Aldehyde Reductase↗

PubMed1994

Ambulatory blood pressure recordings in children and adolescents.

OBJECTIVE: To provide reference data for ambulatory blood pressure monitoring (ABPM) and to determine the influence of age, sex, and race on these values. METHODS: ABPM was performed on 300 healthy, normotensive boys and girls between the ages of 10 and 18 years, including 160 boys and 140 girls, of whom 149 were white and 151 were black. Mean systolic blood pressure (SBP), diastolic blood pressure (DBP), and heart rate (HR) while awake and during sleep were calculated for black and white boys and girls aged 10 to 12 years, 13 to 15 years, and 16 to 18 years. RESULTS: Boys compared with girls 10 to 12 years of age had higher mean (+/- SD) SBP (115 +/- 9 vs 112 +/- 9 mm Hg; P < .01) and DBP (67 +/- 7 vs 65 +/- 5 mm Hg; P < .01) while awake. Boys compared to girls 13 to 15 years of age had higher SBP while awake (116 +/- 11 vs 112 +/- 8 mm Hg; P < .01). Boys compared with girls 16 to 18 years of age had higher SBP while awake (125 +/- 12 vs 111 +/- 9 mm Hg); P < .01) and during sleep (116 +/- 11 vs 106 +/- 9 mm Hg). Comparisons within sex showed similar changes with age for boys and girls. Blacks compared with whites 13 to 15 years of age had higher SBP during sleep (109 +/- 11 vs 105 +/- 10 mm Hg; P < .01), and blacks compared with whites 16 to 18 years of age had higher DBP during sleep (66 +/- 7 vs 58 +/- 6 mm Hg; P < .01). Comparisons across age groups within race showed that blacks 16 to 18 years of age had higher SBP during sleep than blacks 10 to 12 years of age (109 +/- 11 vs 104 +/- 10 mm Hg), and higher DBP during sleep (66 +/- 7 mm Hg; P < .01) than blacks 10 to 12 years of age (61 +/- 7 mm Hg; P < .01) and 13 to 15 years of age (61 +/- 8; P < .01 mm Hg). The changes with age were not significant for white subjects. CONCLUSION: These results provide age-specific reference data for ABPM in youths. These values differ by sex (boys more than girls) and race (Blacks more than Whites).

Adolescent↗

PubMed1993

Probing the active site of human aldose reductase. Site-directed mutagenesis of Asp-43, Tyr-48, Lys-77, and His-110.

Structural models of human aldose reductase complexed with NADPH have revealed the apposition of C4 of the nicotinamide ring with tyrosine 48 and histidine 110, suggesting that either of these residues could function as the proton donor in the reaction mechanism. Tyrosine 48 is also part of a hydrogen-bonding network that includes lysine 77 and aspartate 43. In order to study the potential catalytic roles of these 4 residues, we evaluated the kinetic properties of mutants containing structurally conservative replacements at these sites. Enzymatic activity was undetectable when Tyr-48 was mutated to phenylalanine (Y48F) although affinity for NADPH was unchanged. In contrast, a mutant containing asparagine substituted for His-110 (H110N) was characterized by an almost 80,000-fold increase in Km, but only about a 14-fold reduction in kcat measured with D-glyceraldehyde. Modest changes in catalytic properties were observed in the mutant containing aspartate 43 substituted with asparagine (D43N): Km for aldehyde substrates was elevated up to 17-fold, and kcat decreased less than 16-fold. However, the Kd(NADP) values for D43N were about 5 times higher than those for wild type. Mutant enzyme containing methionine substituted for lysine 77 (K77M) was up to 1,460-fold less active than the wild type. These results are consistent with Tyr-48 acting as the acid-base catalyst in human aldose reductase and confirm the importance of Asp-43, Lys-77, and His-110 to the structure and function of the active site.

Aldehyde Reductase↗

PubMed1993

Refined 1.8 A structure of human aldose reductase complexed with the potent inhibitor zopolrestat.

As the action of aldose reductase (EC 1.1.1.21) is believed to be linked to the pathogenesis of diabetic complications affecting the nervous, renal, and visual systems, the development of therapeutic agents has attracted intense effort. We report the refined 1.8 A x-ray structure of the human holoenzyme complexed with zopolrestat, one of the most potent noncompetitive inhibitors. The zopolrestat fits snugly in the hydrophobic active site pocket and induces a hinge-flap motion of two peptide segments that closes the pocket. Excellent complementarity and affinity are achieved on inhibitor binding by the formation of 110 contacts (< or = 4 A) with 15 residues (10 hydrophobic), 13 with the NADPH coenzyme and 9 with four water molecules. The structure is key to understanding the mode of action of this class of inhibitors and for rational design of better therapeutics.

Aldehyde Reductase↗

PubMed1993

A pre-transition-state mimic of an enzyme: X-ray structure of adenosine deaminase with bound 1-deazaadenosine and zinc-activated water.

The refined 2.4-A structure of adenosine deaminase, recently discovered to be a zinc metalloenzyme [Wilson, D. K., Rudolph, F. B., & Quiocho, F. A. (1991) Science 252, 1278-1284], complexed with the ground-state analog 1-deazaadenosine shows the mode of binding of the analog and, unexpectedly, a zinc-activated water (hydroxide). This structure of a pre-transition-state mimic, combined with that previously determined for the complex with 6(R)-hydroxy-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, sheds new understanding of the precise stereospecificity and hydrolytic catalysis of an important and well-characterized member of a large group of zinc metalloenzymes. As both of these excellent mimics were generated in the active site, they demonstrate a powerful means of dissecting the course of an enzymatic reaction by direct crystallographic analysis.

Adenosine Deaminase↗

PubMed1992

Refined 2.5 A structure of murine adenosine deaminase at pH 6.0.

The X-ray structure of murine adenosine deaminase complexed with the transition-state analogue 6-hydroxyl-1,6-dihydropurine ribonucleoside has been determined from a single crystal grown at pH 4.2 and transferred to mother liquor of increasing pH up to a final pH of 6.0 prior to data collection. The structure has been refined to 2.5 A to a final crystallographic R-factor of 20% using phases from the previously refined 2.4 A structure at pH 4.2. Kinetic measurements show that the enzyme is only 20% active at pH 4.2 whereas it is fully active between pH 6.0 and pH 8.5. The refined structures at either pH are essentially the same. Consideration of the pKa values of the key catalytic residues and the mechanism proposed on the basis of the structure suggests that the ionization state of these residues is largely responsible for the pH dependence on activity.

Adenosine Deaminase↗

PubMed1992

An unlikely sugar substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications.

Aldose reductase, which catalyzes the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of a wide variety of aromatic and aliphatic carbonyl compounds, is implicated in the development of diabetic and galactosemic complications involving the lens, retina, nerves, and kidney. A 1.65 angstrom refined structure of a recombinant human placenta aldose reductase reveals that the enzyme contains a parallel beta 8/alpha 8-barrel motif and establishes a new motif for NADP-binding oxidoreductases. The substrate-binding site is located in a large, deep elliptical pocket at the COOH-terminal end of the beta barrel with a bound NADPH in an extended conformation. The highly hydrophobic nature of the active site pocket greatly favors aromatic and apolar substrates over highly polar monosaccharides. The structure should allow for the rational design of specific inhibitors that might provide molecular understanding of the catalytic mechanism, as well as possible therapeutic agents.

Aldehyde Reductase↗

PubMed1992

A prospective study of familial aggregation of blood pressure in young children.

In a prospective study, we evaluated familial aggregation of blood pressure in a sample of 175 normotensive families with children 3 to 6 yr old. Systolic and diastolic blood pressure measurements of parents and children were correlated at 1, 2, and 3 yr intervals. Spearman rank-order correlation coefficients between parents and children were significant for mother-son pairs. In particular, mother-son blood pressure correlation coefficients were significant for systolic blood pressure across all 3 yr and for diastolic blood pressure during yr 2. Further analyses were performed adjusting for body mass index (BMI), age, physical activity, sodium intake, potassium intake, and parental smoking status, and alcohol use. The Spearman correlation coefficients for mother-son pairs remained significant for yr 1 and 3 after adjusting for these blood pressure correlates. These results are consistent with cross-sectional studies and suggest that both genetic and environmental contributions to blood pressure status are important in young children.

Adult↗

PubMed1992

Weight-for-height measurement and saturated fatty acid intake are predictors of serum cholesterol level in children.

To assess the relationship of serum cholesterol level to anthropometric measurements and dietary intake, we measured serum cholesterol, height, weight, triceps skinfold, and 24-hour dietary intake in 80 children (mean age = 9.8 years) during April 1989. The mean serum cholesterol level was 3.95 mmol/l. In comparison with national data, weight-for-height and triceps skinfold measurements exceeded the 90th percentile in 18 (23%) and 26 (33%) of the children, respectively. Mean cholesterol and fat intakes were 114 mg/1,000 kcal and 36% of energy, respectively. In multiple stepwise regression analyses, weight-for-height measurement and saturated fatty acid intake were most predictive of serum cholesterol levels. Diets of children who consumed 30% of energy or less from fat (low-fat group) met or exceeded the Recommended Dietary Allowances except for energy and vitamin E and were higher in percentage of energy from carbohydrate, dietary fiber, magnesium, iron, and copper than were diets of children who consumed 31% of energy or more from fat (high-fat group). Children in the high-fat group ate more red/processed meats, baked desserts, and fats/oils than children in the low-fat group. Our data indicate that programs to reduce risk of cardiovascular disease in children may need to focus on maintaining ideal body weight and reducing saturated fatty acid intake.

Anthropometry↗

PubMed1991

Atomic structure of adenosine deaminase complexed with a transition-state analog: understanding catalysis and immunodeficiency mutations.

The crystal structure of a murine adenosine deaminase complexed with 6-hydroxyl-1,6-dihydropurine ribonucleoside, a nearly ideal transition-state analog, has been determined and refined at 2.4 angstrom resolution. The structure is folded as an eight-stranded parallel alpha/beta barrel with a deep pocket at the beta-barrel COOH-terminal end wherein the inhibitor and a zinc are bound and completely sequestered. The presence of the zinc cofactor and the precise structure of the bound analog were not previously known. The 6R isomer of the analog is very tightly held in place by the coordination of the 6-hydroxyl to the zinc and the formation of nine hydrogen bonds. On the basis of the structure of the complex a stereoselective addition-elimination or SN2 mechanism of the enzyme is proposed with the zinc atom and the Glu and Asp residues playing key roles. A molecular explanation of a hereditary disease caused by several point mutations of an enzyme is also presented.

Adenosine Deaminase↗