Converting human carbonic anhydrase II into a benzoate ester hydrolase through rational redesign.

Enzymes capable of benzoate ester hydrolysis have several potential medical and industrial applications. A variant of human carbonic anhydrase II (HCAII) was constructed, by rational design, that is capable of hydrolysing para-nitrophenyl benzoate (pNPBenzo) with an efficiency comparable to some naturally occurring esterases. The design was based on a previously developed strategy [G. Höst, L.G. Mårtensson, B.H. Jonsson, Redesign of human carbonic anhydrase II for increased esterase activity and specificity towards esters with long acyl chains, Biochim. Biophys. Acta 1764 (2006) 1601-1606.], in which docking of a transition state analogue (TSA) to the active site of HCAII was used to predict mutations that would allow the reaction. A triple mutant, V121A/V143A/T200A, was thus constructed and shown to hydrolyze pNPBenzo with k(cat)/K(M)=625 (+/- 38) M(-1) s(-1). It is highly active with other ester substrates as well, and hydrolyzes para-nitrophenyl acetate with k(cat)/K(M)=101,700 (+/- 4800) M(-1) s(-1), which is the highest esterase efficiency so far for any CA variant. A parent mutant (V121A/V143A) has measurable K(M) values for para-nitrophenyl butyrate (pNPB) and valerate (pNPV), but for V121A/V143A/T200A no K(M) could be determined, showing that the additional T200A mutation has caused a decreased substrate binding. However, k(cat)/K(M) is higher with both substrates for the triple mutant, indicating that binding energy has been diverted from substrate binding to transition state stabilization.

Experiences among children and adolescents of living with spina bifida and their visions of the future.

Purpose Transitioning to independence may be problematic for persons with spina bifida (SB). Experiences of young persons with SB may provide insights into this group's needs for support. Therefore, the aim of this study was to investigate children's and adolescents' experiences of living with SB, their social and emotional adjustment, and their thoughts about becoming independent adults. Method Semi-structured interviews were conducted with young persons with SB (N = 8, age range 10-17 years). Social and emotional problems were assessed using Beck Youth Inventories. The interview transcripts were analyzed using qualitative content analysis. Results Three main themes were found: being a person with SB; everyday living as a person with SB; and preparing for life as an adult with SB. Indications of emotional and social problems were most prominent among participants with milder physical disability. Conclusions The findings indicate that young persons with SB may overestimate their independence. Other potentially problematic areas were lack of motivation, planning and preparedness for becoming independent. Research on transition to independence in this group should consider assistance at an early age in planning and executing strategies for independence. In addition, the potentially difficult situation for young persons with mild SB should be investigated further. Implications for rehabilitation Children and adolescents with spina bifida might lack the motivation and skills for planning their future and health care programmes should therefore include preparations for life as an adult. Clinical assessment of the level of independence should specifically distinguish between the activities they know how to do and what tasks they actually execute independently. Routine follow-ups should include screening for problems with social and emotional adjustment. Be aware of a potential condition-severity paradox whereby those with less severe impairment might have an increased risk of developing symptoms of social and emotional problems.

Student learning about biomolecular self-assembly using two different external representations.

Self-assembly is the fundamental but counterintuitive principle that explains how ordered biomolecular complexes form spontaneously in the cell. This study investigated the impact of using two external representations of virus self-assembly, an interactive tangible three-dimensional model and a static two-dimensional image, on student learning about the process of self-assembly in a group exercise. A conceptual analysis of self-assembly into a set of facets was performed to support study design and analysis. Written responses were collected in a pretest/posttest experimental design with 32 Swedish university students. A quantitative analysis of close-ended items indicated that the students improved their scores between pretest and posttest, with no significant difference between the conditions (tangible model/image). A qualitative analysis of an open-ended item indicated students were unfamiliar with self-assembly prior to the study. Students in the tangible model condition used the facets of self-assembly in their open-ended posttest responses more frequently than students in the image condition. In particular, it appears that the dynamic properties of the tangible model may support student understanding of self-assembly in terms of the random and reversible nature of molecular interactions. A tentative difference was observed in response complexity, with more multifaceted responses in the tangible model condition.

Combined enzyme and substrate design: grafting of a cooperative two-histidine catalytic motif into a protein targeted at the scissile bond in a designed ester substrate.

A histidine-based, two-residue reactive site for the catalysis of hydrolysis of designed sulfonamide-containing para-nitrophenyl esters has been engineered into a scaffold protein. A matching substrate was designed to exploit the natural active site of human carbonic anhydrase II (HCAII) for well-defined binding. In this we took advantage of the high affinity between the active site zinc atom and sulfonamides. The ester substrate was designed to position the scissile bond in close proximity to the His64 residue in the scaffold protein. Three potential sites for grafting the catalytic His-His pair were identified, and the corresponding N62H/H64, F131H/V135H and L198H/P202H mutants were constructed. The most efficient variant, F131H/V135H, has a maximum k(cat)/K(M) value of approximately 14 000 M(-1) s(-1), with a k(cat) value that is increased by a factor of 3 relative to that of the wild-type HCAII, and by a factor of over 13 relative to the H64A mutant. The results show that an esterase can be designed in a stepwise way by a combination of substrate design and grafting of a designed catalytic motif into a well-defined substrate binding site.