title: The activity and dynamics of enzymes

authors: Dunn, Rachel V.

abstract: It is generally accepted that enzymes require internal flexibility, or the activation of anharmonic motions, for catalytic activity. However, in general, the timescales and forms of the functionally important motions coupled to progress along the reaction pathway remain poorly understood. A number of biophysical studies have shown that protein dynamics undergo a temperature-dependent transition from harmonic to anharmonic motion. The purpose of this study was to further investigate the relationship between protein dynamics and catalytic activity, and also to investigate the nature of the observed dynamic transition.

The activity and dynamics of the single subunit enzyme xylanase, were measured under similar conditions, from –70 to +10⁰C. The activation of anharmonic picosecond-timescale motions is seen above –50⁰C, whereas the activity was seen to follow Arrhenius behaviour over the entire temperature range investigated. This result suggests that the enzyme rate-limiting step is independent of fast anharmonic motions below –50⁰C. Cryoenzymology studies of the temperature dependence of catalase and alkaline phosphatase activity showed no deviations from Arrhenius behaviour down to temperatures near –100⁰C. These results, as well as earlier studies on glutamate dehydrogenase, indicate that the observed independence of low temperature activity on global anharmonic picosecond­timescale motion may be a general property of both single- and multi-subunit enzymes.
Characterisation of cryosolvent properties, such as viscosity and phase changes, by DSC, DMTA, and X-ray scattering techniques, was used to select cryosolvents suitable for low temperature dynamic and activity measurements. A cryosolvent consisting of 70%methanol/10% ethylene glycol/20% water was particularly ideal, as it was the least viscous and free of phase-changes down to at least –160⁰C.

The effect of cryosolvents on enzyme properties was investigated, to enable the effect of solvent and temperature on the enzyme to be distinguished. The effect of varying solution composition on the picosecond timescale dynamics of xylanase was investigated by dynamic neutron scattering. The results indicate a significant effect of the solvent, as the picosecond fluctuations of the protein solution largely follow that of the corresponding pure solvent. The results also indicate that the picosecond-timescale atomic motions respond strongly to melting of pure water, but are relatively invariant in cryosolvents of differing compositions and melting points.

The temperature dependence of the dynamic transition observed for glutamate dehydrogenase-cryosolvent solutions was also determined. Dynamic neutron scattering experiments were performed with two instruments of different energy resolutions, allowing the separate determination of the average dynamical mean-square displacements on timescales of up to approximately 100 ps and 5 ns. The results showed a significant dependence on the timescale of the temperature profile of the mean-square displacement. The lowest temperature at which anharmonic motion is observed is dependent on the time window of the instrument used to observe the dynamics. These results suggest that the temperature dependence of the dynamic transition in average protein motions is timescale dependent. A possible explanation is that motions over a given timescale are progressively replaced by slower motions, as the temperature is reduced.

text: The activity and dynamics of enzymes It is generally accepted that enzymes require internal flexibility, or the activation of anharmonic motions, for catalytic activity. However, in general, the timescales and forms of the functionally important motions coupled to progress along the reaction pathway remain poorly understood. A number of biophysical studies have shown that protein dynamics undergo a temperature-dependent transition from harmonic to anharmonic motion. The purpose of this study was to further investigate the relationship between protein dynamics and catalytic activity, and also to investigate the nature of the observed dynamic transition.

The activity and dynamics of the single subunit enzyme xylanase, were measured under similar conditions, from –70 to +10⁰C. The activation of anharmonic picosecond-timescale motions is seen above –50⁰C, whereas the activity was seen to follow Arrhenius behaviour over the entire temperature range investigated. This result suggests that the enzyme rate-limiting step is independent of fast anharmonic motions below –50⁰C. Cryoenzymology studies of the temperature dependence of catalase and alkaline phosphatase activity showed no deviations from Arrhenius behaviour down to temperatures near –100⁰C. These results, as well as earlier studies on glutamate dehydrogenase, indicate that the observed independence of low temperature activity on global anharmonic picosecond­timescale motion may be a general property of both single- and multi-subunit enzymes.
Characterisation of cryosolvent properties, such as viscosity and phase changes, by DSC, DMTA, and X-ray scattering techniques, was used to select cryosolvents suitable for low temperature dynamic and activity measurements. A cryosolvent consisting of 70%methanol/10% ethylene glycol/20% water was particularly ideal, as it was the least viscous and free of phase-changes down to at least –160⁰C.

The effect of cryosolvents on enzyme properties was investigated, to enable the effect of solvent and temperature on the enzyme to be distinguished. The effect of varying solution composition on the picosecond timescale dynamics of xylanase was investigated by dynamic neutron scattering. The results indicate a significant effect of the solvent, as the picosecond fluctuations of the protein solution largely follow that of the corresponding pure solvent. The results also indicate that the picosecond-timescale atomic motions respond strongly to melting of pure water, but are relatively invariant in cryosolvents of differing compositions and melting points.

The temperature dependence of the dynamic transition observed for glutamate dehydrogenase-cryosolvent solutions was also determined. Dynamic neutron scattering experiments were performed with two instruments of different energy resolutions, allowing the separate determination of the average dynamical mean-square displacements on timescales of up to approximately 100 ps and 5 ns. The results showed a significant dependence on the timescale of the temperature profile of the mean-square displacement. The lowest temperature at which anharmonic motion is observed is dependent on the time window of the instrument used to observe the dynamics. These results suggest that the temperature dependence of the dynamic transition in average protein motions is timescale dependent. A possible explanation is that motions over a given timescale are progressively replaced by slower motions, as the temperature is reduced.

year: 2002

title: "Internet universality": Human rights and principles for the internet

authors: Peters, Michael A.

subjects: Internet

abstract: This paper details proposals by UNESCO to manufacture and draft a concept of “Internet Universality” that adopts a human-rights framework as a basis for articulating a set of principles and rights for the Internet. The paper discusses various drafts of this concept before examining the Charter of Human Rights and Principles for the Internet put forward by The Internet Rights & Principles Dynamic Coalition based at the UN Internet Governance Forum, and the working law Marco Civil da Internet introduced by Brazil.

text: "Internet universality": Human rights and principles for the internet This paper details proposals by UNESCO to manufacture and draft a concept of “Internet Universality” that adopts a human-rights framework as a basis for articulating a set of principles and rights for the Internet. The paper discusses various drafts of this concept before examining the Charter of Human Rights and Principles for the Internet put forward by The Internet Rights & Principles Dynamic Coalition based at the UN Internet Governance Forum, and the working law Marco Civil da Internet introduced by Brazil.

year: 2014

title: The Politics of Knowledge and the Reciprocity Gap in the Governance of Intellectual Property Rights

authors: Emett, Raewyn Anne

subjects: Knowledge

abstract: ABSTRACT
This study examines the politics of knowledge benefit-sharing within the re-regulatory framework of the Trade-related Intellectual Property Rights (TRIPS) Agreement which entered into force in 1995 under the auspices of the World Trade Organisation (WTO). The thesis argues that TRIPS both represents a mainstream legal mechanism for states and organisations to govern ideas through trade, and is characterised by a commercial direction away from multilateralism to bilateralism. In its post-implementation phase, this situation has seen the strongest states and corporations consolidate extensive markets in knowledge goods and services. Through analyses of the various levels of international and national governance within the competitive knowledge structure of international political economy (IPE), this study argues that the politicisation of intellectual property has resulted in the dislocation of reciprocity from its normative roots in fairness and trade equity. In conducting this enquiry the research focuses on the political manifestations of intellectual property consistent with long-standing epistemic considerations of reciprocity to test the extent to which the intrinsic public good value of knowledge and its importance to human societies can be reconciled with the privatisation of public forms of knowledge related to discoveries and innovations. 

This thesis draws on Becker's virtue-theoretic model of reciprocity premised on normative obligations to social life to ground its claim that an absence of substantive reciprocal requirements capable of sustaining equivalent returns and rewards is detrimental, both theoretically and practically, to the intrinsic socio-cultural foundation and public good value of knowledge. The conceptual framework of reciprocity defined and developed in this study challenges the materialist controlling authority and proprietary ownership vested in intellectual property law. A new conceptual approach proposed through reciprocity, and provoked by on-going debates about IP recognition, knowledge protection, access and distribution is advanced to counter strengthened and expanded IPRs. Theories of knowledge and property drawn from political philosophies are employed to test whether reciprocity is sufficiently robust enough, or even capable of, encompassing the gap between capital and applied science. This thesis argues that hyper-capitalism at global, national and local levels, accompanied by the boundless accumulation of technology, closes down competition both compromising IP as private rights and the viability of their governance. The political implications of the protection and enforcement of private rights through IP is examined in two key chapters utilising empirical data in relation to traditional knowledge (TK) and reciprocity; the first sets the parameters of TK and the second explores aspects of Māori knowledge systems and reciprocity directed at identifying national and local issues of significance to the debates on IP governance. As a viable direction for knowledge governance this thesis concludes that the gap between the re-regulatory trade framework of intellectual property on the one hand, and reciprocity on the other, requires closing to ameliorate the detrimental disruptions to democratic integrity, fairness and trade equity for significant numbers of communities and peoples around the world.

text: The Politics of Knowledge and the Reciprocity Gap in the Governance of Intellectual Property Rights ABSTRACT
This study examines the politics of knowledge benefit-sharing within the re-regulatory framework of the Trade-related Intellectual Property Rights (TRIPS) Agreement which entered into force in 1995 under the auspices of the World Trade Organisation (WTO). The thesis argues that TRIPS both represents a mainstream legal mechanism for states and organisations to govern ideas through trade, and is characterised by a commercial direction away from multilateralism to bilateralism. In its post-implementation phase, this situation has seen the strongest states and corporations consolidate extensive markets in knowledge goods and services. Through analyses of the various levels of international and national governance within the competitive knowledge structure of international political economy (IPE), this study argues that the politicisation of intellectual property has resulted in the dislocation of reciprocity from its normative roots in fairness and trade equity. In conducting this enquiry the research focuses on the political manifestations of intellectual property consistent with long-standing epistemic considerations of reciprocity to test the extent to which the intrinsic public good value of knowledge and its importance to human societies can be reconciled with the privatisation of public forms of knowledge related to discoveries and innovations. 

This thesis draws on Becker's virtue-theoretic model of reciprocity premised on normative obligations to social life to ground its claim that an absence of substantive reciprocal requirements capable of sustaining equivalent returns and rewards is detrimental, both theoretically and practically, to the intrinsic socio-cultural foundation and public good value of knowledge. The conceptual framework of reciprocity defined and developed in this study challenges the materialist controlling authority and proprietary ownership vested in intellectual property law. A new conceptual approach proposed through reciprocity, and provoked by on-going debates about IP recognition, knowledge protection, access and distribution is advanced to counter strengthened and expanded IPRs. Theories of knowledge and property drawn from political philosophies are employed to test whether reciprocity is sufficiently robust enough, or even capable of, encompassing the gap between capital and applied science. This thesis argues that hyper-capitalism at global, national and local levels, accompanied by the boundless accumulation of technology, closes down competition both compromising IP as private rights and the viability of their governance. The political implications of the protection and enforcement of private rights through IP is examined in two key chapters utilising empirical data in relation to traditional knowledge (TK) and reciprocity; the first sets the parameters of TK and the second explores aspects of Māori knowledge systems and reciprocity directed at identifying national and local issues of significance to the debates on IP governance. As a viable direction for knowledge governance this thesis concludes that the gap between the re-regulatory trade framework of intellectual property on the one hand, and reciprocity on the other, requires closing to ameliorate the detrimental disruptions to democratic integrity, fairness and trade equity for significant numbers of communities and peoples around the world.

year: 2007