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L Ridder

Publications and source records attributed to L Ridder.

5 recordsLinked to original sources
PubMed2000

Modelling flavin and substrate substituent effects on the activation barrier and rate of oxygen transfer by p-hydroxybenzoate hydroxylase.

The simulation of enzymatic reactions, using computer models, is becoming a powerful tool in the most fundamental challenge in biochemistry: to relate the catalytic activity of enzymes to their structure. In the present study, various computed parameters were correlated with the natural logarithm of experimental rate constants for the hydroxylation of various substrate derivatives catalysed by wild-type para-hydroxybenzoate hydroxylase (PHBH) as well as for the hydroxylation of the native substrate (p-hydroxybenzoate) by PHBH reconstituted with a series of 8-substituted flavins. The following relative parameters have been calculated and tested: (a) energy barriers from combined quantum mechanical/molecular mechanical (QM/MM) (AM1/CHARMM) reaction pathway calculations, (b) gas-phase reaction enthalpies (AM1) and (c) differences between the HOMO and LUMO energies of the isolated substrate and cofactor molecules (AM1 and B3LYP/6-31+G(d)). The gas-phase approaches yielded good correlations, as long as similarly charged species are involved. The QM/MM approach resulted in a good correlation, even including differently charged species. This indicates that the QM/MM model accounts quite well for the solvation effects of the active site surroundings, which vary for differently charged species. The correlations obtained demonstrate quantitative structure activity relationships for an enzyme-catalysed reaction including, for the first time, substitutions on both substrate and cofactor.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1998

Quantitative structure/activity relationship for the rate of conversion of C4-substituted catechols by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1.

The influence of various C4/C5 substituents in catechol (1,2-dihydroxybenzene) derivatives on the overall rate of conversion by catechol-1,2-dioxygenase from Pseudomonas putida (arvilla) C1 was investigated. Using catechol, 4-methylcatechol, 4-fluorocatechol, 4-chlorocatechol, 4-bromocatechol, 4,5-difluorocatechol and 4-chloro-5-fluorocatechol, it could be demonstrated that substituents at the C4 and/or C5 position decrease the rate of conversion, from 62% (4-methylcatechol) down to 0.7% (4-chloro-5-fluorocatechol) of the activity with non-substituted catechol. The inhibition was reversible upon addition of excess catechol for all substrates tested. This indicates that the lower activities are neither due to irreversible inactivation of the enzyme nor to product inhibition. Based on the reaction mechanism proposed in the literature [Que, L. & Ho, R. Y. N. (1996) Chem. Rev. 96, 2606-2624], the nucleophilic reactivity of the catecholate was expected to be an essential characteristic for its conversion by catechol-1,2-dioxygenase. Therefore, the rates of conversion were compared with calculated energies of the highest occupied molecular orbital (E(HOMO)) of the substrates. A clear quantitative relationship (R>0.97) between the ln kcat and the calculated electronic parameter E(HOMO) was obtained. This indicates that the rate-limiting step of the reaction cycle is dependent on the nucleophilic reactivity of the substrate and not sterically hindered by the relatively large bromine or methyl substituents used in the present study. Possible steps in the reaction mechanism determining the overall rate at 20 degrees C are discussed.

Catechol 1,2-Dioxygenase↗
PubMedDate not supplied

Combined quantum mechanical and molecular mechanical reaction pathway calculation for aromatic hydroxylation by p-hydroxybenzoate-3-hydroxylase.

The reaction pathway for the aromatic 3-hydroxylation of p-hydroxybenzoate by the reactive C4a-hydroperoxyflavin cofactor intermediate in p-hydroxybenzoate hydroxylase (PHBH) has been investigated by a combined quantum mechanical and molecular mechanical (QM/MM) method. A structural model for the C4a-hydroperoxyflavin intermediate in the PHBH reaction cycle was built on the basis of the crystal structure coordinates of the enzyme-substrate complex. A reaction pathway for the subsequent hydroxylation step was calculated by imposing a reaction coordinate that involves cleavage of the peroxide oxygen-oxygen bond and formation of the carbon-oxygen bond between the C3 atom of the substrate and the distal oxygen of the peroxide moiety of the cofactor. The geometric changes and the Mulliken charge distributions along the calculated reaction pathway are in line with an electrophilic aromatic substitution type of mechanism. The energy barrier of the calculated reaction is considerably lower when the substrate hydroxyl moiety is deprotonated, in comparison with the barrier found with a protonated hydroxyl moiety. This effect of the protonation state of the substrate on the calculated energy barrier supports experimental observations that deprotonation is required for hydroxylation of the substrate. A notable event in the calculated reaction pathway is a lengthening of the peroxide oxygen-oxygen bond at an intermediate stage. Further analysis of the reaction pathway indicates that this oxygen-oxygen bond elongation is accompanied by an increase in electrophilic reactivity on the distal oxygen of the peroxide moiety, which may assist the C-O bond formation in the reaction of the C4a-hydroperoxyflavin intermediate with the substrate. Analysis of the effect of individual active site residues on the reaction reveals a specific transition state stabilization by the backbone carbonyl moiety of Pro293. The crystal water 717 appears to drive the hydroxylation step through a stabilizing hydrogen bond interaction to the proximal oxygen of the C4a-hydroperoxyflavin intermediate, which increases in strength as the hydroperoxyflavin cofactor converts to the anionic (deprotonated) hydroxyflavin.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMedDate not supplied

Outcomes of a nursing intervention for siblings of chronically ill children: a pilot study.

PURPOSE: To evaluate the outcomes of a structured, educational, and support group intervention (ISEE, Intervention for Siblings: Experience Enhancement) for siblings of children with chronic illness (cancer, cystic fibrosis, diabetes, and spina bifida), including a session with parents about sibling needs; and to describe sibling and parent perceptions of sibling experiences at home. DESIGN: One-group, pretest-posttest pilot study. PARTICIPANTS: A convenience sample of 22 siblings and parents. SETTING: A Midwestern university medical center. MAIN OUTCOME MEASURES: Knowledge of Illness Test, parent ratings on a global, single item, 10-point scale. RESULTS: Sibling test scores increased significantly after intervention, compared to baseline. Parents' average evaluation rating was 9 on a 10-point scale. Parents supported their positive ratings with verbatim descriptions. Sibling and parent perceptions of sibling experiences were congruent, suggesting the sources of potential adjustment problems in siblings, and were consistent with the literature. CONCLUSIONS: A randomized, clinical trial with a larger sample size is needed to evaluate the intervention further.

Adolescent↗