title: Heat capacity changes for transition-state analogue binding and catalysis with human 5 '-methylthioadenosine phosphorylase

authors: Firestone, Ross S.

subjects: Science & Technology

abstract: Human 5′-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5′-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (−ΔCₚ), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCₚ of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCₚ. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCₚ for binding. In addition, ΔCₚ values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (−ΔCₚ‡). A comparison of the ΔCₚ‡ for MTAP presteady state chemistry and ΔCₚ for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP–inhibitor complexes indicate remarkable protein thermal stability (to Tₘ = 99 °C) in complexes with transition-state analogues.

text: Heat capacity changes for transition-state analogue binding and catalysis with human 5 '-methylthioadenosine phosphorylase Human 5′-methylthioadenosine phosphorylase (MTAP) catalyzes the phosphorolysis of 5′-methylthioadenosine (MTA). Its action regulates cellular MTA and links polyamine synthesis to S-adenosylmethionine (AdoMet) salvage. Transition state analogues with picomolar dissociation constants bind to MTAP in an entropically driven process at physiological temperatures, suggesting increased hydrophobic character or dynamic structure for the complexes. Inhibitor binding exhibits a negative heat capacity change (−ΔCₚ), and thus the changes in enthalpy and entropy upon binding are strongly temperature-dependent. The ΔCₚ of inhibitor binding by isothermal titration calorimetry does not follow conventional trends and is contrary to that expected from the hydrophobic effect. Thus, ligands of increasing hydrophobicity bind with increasing values of ΔCₚ. Crystal structures of MTAP complexed to transition-state analogues MT-DADMe-ImmA, BT-DADMe-ImmA, PrT-ImmA, and a substrate analogue, MT-tubercidin, reveal similar active site contacts and overall protein structural parameters, despite large differences in ΔCₚ for binding. In addition, ΔCₚ values are not correlated with Kd values. Temperature dependence of presteady state kinetics revealed the chemical step for the MTAP reaction to have a negative heat capacity for transition state formation (−ΔCₚ‡). A comparison of the ΔCₚ‡ for MTAP presteady state chemistry and ΔCₚ for inhibitor binding revealed those transition-state analogues most structurally and thermodynamically similar to the transition state. Molecular dynamics simulations of MTAP apoenzyme and complexes with MT-DADMe-ImmA and MT-tubercidin show small, but increased dynamic motion in the inhibited complexes. Variable temperature CD spectroscopy studies for MTAP–inhibitor complexes indicate remarkable protein thermal stability (to Tₘ = 99 °C) in complexes with transition-state analogues.

year: 2017

title: Determination of EDTA in dairy wastewater and adjacent surface water

authors: Xie, Congmin Zoe

subjects: dairy wastewater

abstract: An HPLC-UV analytical method was developed to determine ethylenediaminetetraacetic acid (EDTA) in dairy wastewater and surface water. The optimizing separation was achieved by reversed–phase ion-pair liquid chromatography on a C18 column using methanol as mobile phase solvent, tetrabutylammonium bromide as the ion-pair reagent in pH 3.3 formate buffer solution at a flow rate of 0.9 mL min⁻¹ with a UV detector at 265 nm. No interference of Ca, Mg or NO₃⁻ was detected. Method performance was evaluated in terms of linearity, repeatability and reproducibility. The method detection limit was 5 µg L⁻¹. The contents of EDTA in dairy effluents were 72 ~ 261 µg L⁻¹ at a large dairy site. A change of EDTA concentration was observed downstream of the dairy effluent discharge, but this was well under the predicted no effect concentration for aquatic ecosystem

text: Determination of EDTA in dairy wastewater and adjacent surface water An HPLC-UV analytical method was developed to determine ethylenediaminetetraacetic acid (EDTA) in dairy wastewater and surface water. The optimizing separation was achieved by reversed–phase ion-pair liquid chromatography on a C18 column using methanol as mobile phase solvent, tetrabutylammonium bromide as the ion-pair reagent in pH 3.3 formate buffer solution at a flow rate of 0.9 mL min⁻¹ with a UV detector at 265 nm. No interference of Ca, Mg or NO₃⁻ was detected. Method performance was evaluated in terms of linearity, repeatability and reproducibility. The method detection limit was 5 µg L⁻¹. The contents of EDTA in dairy effluents were 72 ~ 261 µg L⁻¹ at a large dairy site. A change of EDTA concentration was observed downstream of the dairy effluent discharge, but this was well under the predicted no effect concentration for aquatic ecosystem

year: 2010

title: Pilot study investigating pathway for practitioner engagement with whānau during a self-control activity: Implications for uptake in the home and for empowered engagement.

authors: Watson, Kaitlyn

subjects: Community psychology

abstract: There is a gap in youth mentoring services using the home environment of at-risk youth to encourage development of the skills worked on within mentoring. This thesis explores whether a game-based intervention situated in the home environment of youth may initiate changes in behaviour that coincide with their learning during mentoring. Participants were recruited through a private youth mentoring service within Whāngarei. Participants included whānau of youth who are enrolled in the Awhi Ako youth mentoring service and their respective mentors. The Awhi Ako mentoring service aims to support youth through their selected needs by a wider wraparound service.
A game was picked to introduce to whānau to play together at home. This game contained a mechanism that may reward self-control behaviours. Whānau were interviewed and asked to discuss their child’s self-control behaviours before the introduction of the game and any changes after playing the game. 
A principal focus of this thesis is on practitioner engagement with the whānau of mentored youth. Practitioner engagement with communities and people is an integral value underpinning Community psychology. Whānau within the youth mentoring service include Māori, Pasifika, Pākehā and mixed ethnic backgrounds. A core focus of this thesis is understanding the relationship between practitioner and participants and how this impacts upon uptake of the introduction of the activity. In accordance with community psychology underpinnings and values of cultural awareness, cultural respect and understanding this thesis aspires to provide evidence towards the importance of this relationship. Another key focus of this thesis is comprehending the multifaceted effects of an intervention within the home environment and how this impacts mentors, the home environment and the development of youth. Emphasizing a fundamental principle of community psychology of interconnectedness between all facets of a persons life. 
The conclusions drawn from this thesis illustrate the importance of developing a relationship with whānau a key focus to their willingness to participate and actively engage in this pilot study. The findings from this pilot study indicate that whānau experienced hardship and that whānau within at-risk environments had competing priorities that impeded the introduction of home-based activities.

text: Pilot study investigating pathway for practitioner engagement with whānau during a self-control activity: Implications for uptake in the home and for empowered engagement. There is a gap in youth mentoring services using the home environment of at-risk youth to encourage development of the skills worked on within mentoring. This thesis explores whether a game-based intervention situated in the home environment of youth may initiate changes in behaviour that coincide with their learning during mentoring. Participants were recruited through a private youth mentoring service within Whāngarei. Participants included whānau of youth who are enrolled in the Awhi Ako youth mentoring service and their respective mentors. The Awhi Ako mentoring service aims to support youth through their selected needs by a wider wraparound service.
A game was picked to introduce to whānau to play together at home. This game contained a mechanism that may reward self-control behaviours. Whānau were interviewed and asked to discuss their child’s self-control behaviours before the introduction of the game and any changes after playing the game. 
A principal focus of this thesis is on practitioner engagement with the whānau of mentored youth. Practitioner engagement with communities and people is an integral value underpinning Community psychology. Whānau within the youth mentoring service include Māori, Pasifika, Pākehā and mixed ethnic backgrounds. A core focus of this thesis is understanding the relationship between practitioner and participants and how this impacts upon uptake of the introduction of the activity. In accordance with community psychology underpinnings and values of cultural awareness, cultural respect and understanding this thesis aspires to provide evidence towards the importance of this relationship. Another key focus of this thesis is comprehending the multifaceted effects of an intervention within the home environment and how this impacts mentors, the home environment and the development of youth. Emphasizing a fundamental principle of community psychology of interconnectedness between all facets of a persons life. 
The conclusions drawn from this thesis illustrate the importance of developing a relationship with whānau a key focus to their willingness to participate and actively engage in this pilot study. The findings from this pilot study indicate that whānau experienced hardship and that whānau within at-risk environments had competing priorities that impeded the introduction of home-based activities.

year: 2024