title: Decoupling enzyme catalysis from thermal denaturation

authors: Easter, Ashley Davys

subjects: equilibrium model

abstract: The equilibrium model (EM) (Daniel et al., 2001) postulates two forms of a folded enzyme, one catalytically active (Eact) and the other inactive (Einact), which interconvert via a fast thermal equilibrium (Keq) (Figure A). This model for enzyme catalysis accounts for experimentally observed time and temperature profiles of enzyme/substrate systems more accurately than the classically derived, single folded-species, model (Figure B). In both models, the denatured species (X) is formed via the kinact process, which is temperature and time-dependent.
(FIgure A) (Figure B) Comparison between the equilibrium model and classical model for enzyme catalysis. For both, the vertical axis is catalytic rate (M s-1), the left-right axis is increasing temperature (K) and the back-front axis is assay duration (s).
The physical basis for the Eact/Einact equilibrium is unknown. To study the equilibrium, the temperature midpoint of the Eact/Einact transition (Teq) has to be separated from the thermostability of the enzyme (Tm) to allow the Einact species to exist in measurable concentrations without exhibiting denaturation.
Mutations were made in a well-studied and NMR-accessible ribonuclease, barnase, to alter the thermostability and/or the Teq of the enzyme activity. The stability properties of each mutant were measured and the activity against two substrates assayed. New models were derived and fitted against wild-type barnase, and an ideal data set, to give insight into alternative irreversible and reversible denaturation pathways. Simulations of these models were developed to benchmark potential dynamics work and explain the movements of species within each model's framework.
Assay data fits to the EM and alternative models show a preference for irreversible denaturation pathways via the Einact species. A mathematically simplified model was also found that accounts for data and could provide an alternative method for determining EM parameters. Although fits of barnase to the EM were statistically good, the denaturation properties could not be reconciled with the literature or experimentally determined values for stability and unfolding. Simulations illustrating how the Eact, Einact and denatured (X) species interact also corroborate this finding.
Despite this discrepancy (in fitted parameters to the EM), it is hypothesised that the Teq and Tm of a disulphide-bridged mutant of barnase have been successfully decoupled. This mutant has been 15N- labelled for future NMR dynamics measurements. New approaches to the EM model are proposed where the separate determination of enzyme thermodynamic properties (e.g., rate and free energy of denaturation) would allow other EM parameters to be fitted independently to each data set.

text: Decoupling enzyme catalysis from thermal denaturation The equilibrium model (EM) (Daniel et al., 2001) postulates two forms of a folded enzyme, one catalytically active (Eact) and the other inactive (Einact), which interconvert via a fast thermal equilibrium (Keq) (Figure A). This model for enzyme catalysis accounts for experimentally observed time and temperature profiles of enzyme/substrate systems more accurately than the classically derived, single folded-species, model (Figure B). In both models, the denatured species (X) is formed via the kinact process, which is temperature and time-dependent.
(FIgure A) (Figure B) Comparison between the equilibrium model and classical model for enzyme catalysis. For both, the vertical axis is catalytic rate (M s-1), the left-right axis is increasing temperature (K) and the back-front axis is assay duration (s).
The physical basis for the Eact/Einact equilibrium is unknown. To study the equilibrium, the temperature midpoint of the Eact/Einact transition (Teq) has to be separated from the thermostability of the enzyme (Tm) to allow the Einact species to exist in measurable concentrations without exhibiting denaturation.
Mutations were made in a well-studied and NMR-accessible ribonuclease, barnase, to alter the thermostability and/or the Teq of the enzyme activity. The stability properties of each mutant were measured and the activity against two substrates assayed. New models were derived and fitted against wild-type barnase, and an ideal data set, to give insight into alternative irreversible and reversible denaturation pathways. Simulations of these models were developed to benchmark potential dynamics work and explain the movements of species within each model's framework.
Assay data fits to the EM and alternative models show a preference for irreversible denaturation pathways via the Einact species. A mathematically simplified model was also found that accounts for data and could provide an alternative method for determining EM parameters. Although fits of barnase to the EM were statistically good, the denaturation properties could not be reconciled with the literature or experimentally determined values for stability and unfolding. Simulations illustrating how the Eact, Einact and denatured (X) species interact also corroborate this finding.
Despite this discrepancy (in fitted parameters to the EM), it is hypothesised that the Teq and Tm of a disulphide-bridged mutant of barnase have been successfully decoupled. This mutant has been 15N- labelled for future NMR dynamics measurements. New approaches to the EM model are proposed where the separate determination of enzyme thermodynamic properties (e.g., rate and free energy of denaturation) would allow other EM parameters to be fitted independently to each data set.

year: 2010

title: Why unavoidable moral wrongdoing is impossible

authors: Lane, Aaron

subjects: ethics

abstract: We can be faced with situations in which we act in accordance with the all-things-considered morally right course of action, and yet nevertheless experience an emotion of self-directed distress at the fact that we have so acted. Moreover, we have the intuition that this emotion is rational or appropriate. Literature is scattered with such examples, and they crop up not infrequently in life.

The fact that agents experience justified distress in such situations has been taken as evidence that unavoidable moral wrongdoing is possible. However, if this is the case, then the compelling intuition that an all-things-considered morally right action cannot be wrong must be false.

In this thesis, I offer a way of reconciling these two compelling intuitions that denies the possibility of unavoidable moral wrongdoing. I argue that existing attempts to dissolve this contradiction by arguing in favour of the possibility of unavoidable moral wrongdoing are unsatisfactory; and I contend that, by utilising Susan Wolf’s ideas on the “nameless virtue” (2001), developed in order to solve the problem of resultant moral luck, we can arrive at a more compelling method of dissolving the apparent contradiction. This solution is one that allows us to retain the intuition that a right act cannot also be wrong, while simultaneously allowing for the rationality of emotion of self-directed distress agents experience in situations of apparent unavoidable moral wrongdoing.

text: Why unavoidable moral wrongdoing is impossible We can be faced with situations in which we act in accordance with the all-things-considered morally right course of action, and yet nevertheless experience an emotion of self-directed distress at the fact that we have so acted. Moreover, we have the intuition that this emotion is rational or appropriate. Literature is scattered with such examples, and they crop up not infrequently in life.

The fact that agents experience justified distress in such situations has been taken as evidence that unavoidable moral wrongdoing is possible. However, if this is the case, then the compelling intuition that an all-things-considered morally right action cannot be wrong must be false.

In this thesis, I offer a way of reconciling these two compelling intuitions that denies the possibility of unavoidable moral wrongdoing. I argue that existing attempts to dissolve this contradiction by arguing in favour of the possibility of unavoidable moral wrongdoing are unsatisfactory; and I contend that, by utilising Susan Wolf’s ideas on the “nameless virtue” (2001), developed in order to solve the problem of resultant moral luck, we can arrive at a more compelling method of dissolving the apparent contradiction. This solution is one that allows us to retain the intuition that a right act cannot also be wrong, while simultaneously allowing for the rationality of emotion of self-directed distress agents experience in situations of apparent unavoidable moral wrongdoing.

year: 2025-10-17

title: A collaborative approach to improving youth mental health.

authors: Goodwin, Debbie Waireka Tuhi

subjects: Maori

abstract: Nga Taiohi Whai Marama / Youth Connex was developed collaboratively by three organisations: Health Waikato Mental Health, Te Runanga o Kirikiriroa, and Linkage. These organisations had a vision of creating greater integration and improved pathways between youth and mental health services, as well as providing a service explicitly based on youth-friendly principles. The key aims of Whai Marama Youth Connex are to improve youth’s access to services and contribute to the development of a youth service-provider network. To fulfil this we aim to provide a range of services, integrated pathways and capacity building on several levels, the client level, the provider level and the funder level. Debbie Goodwin (Tuhoe) was employed as the Manager of the service and has a key role in developing the organisation, planning and shaping its services, and employing and managing its’ staff. She will give an overview of how the service is developing in partnership and biculturally, how youth-friendly principles are enacted, and what some of the key issues have been in its early stages.

text: A collaborative approach to improving youth mental health. Nga Taiohi Whai Marama / Youth Connex was developed collaboratively by three organisations: Health Waikato Mental Health, Te Runanga o Kirikiriroa, and Linkage. These organisations had a vision of creating greater integration and improved pathways between youth and mental health services, as well as providing a service explicitly based on youth-friendly principles. The key aims of Whai Marama Youth Connex are to improve youth’s access to services and contribute to the development of a youth service-provider network. To fulfil this we aim to provide a range of services, integrated pathways and capacity building on several levels, the client level, the provider level and the funder level. Debbie Goodwin (Tuhoe) was employed as the Manager of the service and has a key role in developing the organisation, planning and shaping its services, and employing and managing its’ staff. She will give an overview of how the service is developing in partnership and biculturally, how youth-friendly principles are enacted, and what some of the key issues have been in its early stages.

year: 2003