The thermal behaviour of enzyme activity: implications for biotechnology

Opinion

TRENDS in Biotechnology

Vol.24 No.7 July 2006

The thermal behaviour of enzyme activity: implications for biotechnology Robert Eisenthal1, Michelle E. Peterson2, Roy M. Daniel2 and Michael J. Danson3 1

Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK Department of Biological Sciences, University of Waikato, Private Bag 3105, Hamilton, New Zealand 3 Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK 2

The way that enzymes respond to temperature is fundamental to many areas of biotechnology. This has long been explained in terms of enzyme stability and catalytic activation energy, but recent observations of enzyme behaviour suggest that this picture is incomplete. We have developed and experimentally validated a new model to describe the effect of temperature on enzymes; this model incorporates additional fundamental parameters that enable a complete description of the effects of temperature on enzyme activity. In this article, we consider the biotechnological implications of this model in the areas of enzyme engineering, enzyme reactor operation and the selection and/or screening of useful enzymes from the environment. Introduction Enzymes with high stability are still a prime target for biotechnology, for their prolonged life in storage and application and for their utility under extreme conditions [1–3]. In addition to withstanding high temperatures, such enzymes are also resistant to organic solvents and proteolytic attack [4,5]. Hyperstable enzymes have been sought from organisms growing in thermal environments, although engineering thermal stability into mesophilic enzymes is an attractive alternative. The increased availability of complete genomes, coupled with the ready resolution of enzyme structures and the ability to manipulate them at will, has provided the biotechnologist with the potential to obtain stable enzymes designed for individual applications. In this respect, both the thermostability of the enzyme and the temperature at which an enzyme is optimally active (Topt) are crucial parameters. In this article, we explore the biotechnological implications of our recent description of a new model for the thermal behaviour of enzymes [6] that is applicable to their isolation, engineering and use. The equilibrium model Traditionally, the dependence of enzyme activity on temperature has been described by the ‘Classical Model’, which consists of two processes: the catalytic reaction (defined by kcat) and irreversible inactivation (defined by kinact). The temperature dependence of these rate constants is characterised, respectively, by the catalytic Corresponding author: Danson, M.J. ([email protected]). Available online 6 June 2006

activation energy, DG*cat, and the activation energy for the thermal inactivation reaction, DG*inact. Consequently, if the rate of enzyme activity during a fixed assay time is plotted against temperature, these two parameters will lead to a temperature optimum that is dependent on the assay duration; that is, because thermal denaturation is time dependent, changing the assay duration changes the apparent optimum temperature, Topt [7,8]. Recent observations show that for some enzymes less activity is observed at high temperatures than can be accounted for by thermal denaturation [9,10]. Moreover, in several cases we have observed that the decrease in enzymic activity at temperatures above Topt is reversible – the enzyme regains its optimal activity as the temperature is decreased [10]. In response to these observations, we have hypothesized that the temperature dependence of enzyme activity is indeed reversible, at least in part, and have developed and validated a model that incorporates the features of both reversibility and time-dependent inactivation [6,11]. This model (the Equilibrium Model) includes an inactive form of the enzyme (Einact) in reversible equilibrium with the active form (Eact); it is the inactive form that undergoes irreversible thermal inactivation to the thermally denatured state (X) (Equation 1): Keq

K inact

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The equilibrium is described by an equilibrium constant (Keq), the temperature dependence of which is characterised in terms of the enthalpy of the equilibrium (DHeq) and a new thermal parameter (Teq), which is the temperature at which the concentrations of Eact and Einact are equal. Thus, TeqZDHeq/DSeq, the enthalpic and entropic changes associated with the interconversion of Eact and Einact; that is, the entropic term is subsumed into the parameter Teq. A useful feature of Teq is that it enables the prediction of Topt, the temperature at which enzyme activity is maximal [11]. Another characteristic of this model is that it ‘buffers’ the enzyme against irreversible inactivation, defined by the inactivation rate constant, Kinact, the value of which is also temperature dependent. The buffering action occurs because only a fraction of the total native enzyme is susceptible to inactivation at any time [6]. Of the 30 or so enzymes we have studied in detail, all follow the Equilibrium Model [11] (M.E. Peterson, C.K. Lee and R.M. Daniel, unpublished data).

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Figure 1. Comparison of the (a) Equilibrium and (b) Classical models of temperature dependence of enzyme activity. The data were simulated using parameter values: (a) DG*inactZ95 kJ molK1, DG*catZ75 kJ molK1, HeqZ95 kJ molK1, TeqZ320 K; (b) DG*inactZ95 kJ molK1, DG*catZ75 kJ molK1. Note the absence of a temperature optimum at zero time in the Classical model.

global-unfolding. Thus, Tm is the generally accepted parameter that defines the thermostability of a protein, as opposed to Teq, which is a major factor influencing the temperature dependence of the catalytic activity of an enzyme. The idea of a temperature-dependent, reversible equilibrium between active and inactive forms of an enzyme was proposed in 1944 by Sizer [13]; however, Sizer’s model did not include the possibility of timedependent, irreversible inactivation. Time-dependent inactivation was considered by Wright and Schomaker [14], who studied the inactivation of diphtheria antitoxin by heat. They proposed a model that comprised two interconvertible forms of active antibody, one susceptible to irreversible inactivation and the other protected. They made no assumption of a rapid equilibrium and treated the system as one consisting of three first-order reactions. They recognized that the model provided a reservoir of native antibody during a significant fraction of the time course. However, their model differs from the Equilibrium

The effect of incorporating an additional inactive species of the enzyme in reversible equilibrium with the active form is most evident from a plot of enzyme activity versus temperature versus time (Figure 1a). This shows a temperature optimum at zero time, which is not seen in the Classical Model (Figure 1b). At temperatures above the maximal enzyme activity, the loss of activity due to the shift in the Eact/Einact equilibrium is effectively instantaneous (!1s) and at least two orders of magnitude faster than denaturation at temperatures above the maximal enzyme activity [11,12]. Furthermore, Teq is essentially unaffected by agents such as betaine and guanidinium hydrochloride at concentrations that respectively enhance and lower the stability of the enzyme (Figure 2); this shows that Teq is independent of thermostability. At this point, it is important to emphasize the difference between Teq, which relates to the reversible equilibrium between Eact and Einact, and Tm, the melting temperature, which is equal to the mid-point of the temperature range over which a protein undergoes thermally induced

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Figure 2. Experimentally determined temperature and time profiles of the enzymic activity of aryl-acylamidase [EC 3.5.1.13] in the presence of (a) 0.1 M guanidine hydrochloride; (b) no addition; and (c) 0.1 M betaine. The enzyme was assayed as described in Peterson et al. [11] The curve traced by the border between the blue- and redshaded areas represents the progress curve of irreversible inactivation at 315 K to highlight the differences in rates of inactivation under the different conditions. www.sciencedirect.com

Opinion

TRENDS in Biotechnology

Model in assuming that the ‘activity’ (which was based on a bioassay) is independent of temperature and that both non-denatured forms are active. Biotechnological implications of the equilibrium model There are three areas where knowledge of Teq might be essential to the optimal use of enzymes in biotechnology: enzyme engineering, enzyme reactor operation and the selection and/or screening of useful enzymes from the environment.

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Indirect methods involving, for example, fluorescence transfer as a measure of conformational change might be applicable if labelling of the active site could be achieved without destruction of its catalytic activity.

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Enzyme engineering The Equilibrium Model introduces an additional and separate factor, Teq, to be considered when trying to enhance enzyme activity at high temperatures. The model tells us that engineering an increase in thermostability alone will not necessarily lead to enhanced activity at high temperatures unless the temperature of the equilibrium mid-point between the active and inactive forms, Teq, is also shifted to higher temperatures. Moreover, if the Teq of the engineered enzyme is unaffected or lowered, and thermostability is assessed by a high-temperature enzyme assay, increased stability might not be detected upon the screening of mutants. This phenomenon is clearly demonstrated in Figure 2, which shows the effect of agents that increase or decrease the thermal stability of arylacylamidase. It should be noted that, although the rate of irreversible inactivation is affected by the presence of guanidine (a denaturing agent) and betaine (a stabilizing agent), the temperature and/or activity profile at time zero is virtually identical to that of the enzyme in buffer alone. The activity of the enzyme at the temperature optimum at time zero is similarly unaffected. The molecular basis of the equilibrium described by Teq has not been defined; however, because it is unaffected by denaturing or stabilizing agents but is affected by changes in substrate (M.E. Peterson, PhD thesis, University of Waikato, New Zealand, 2005), it is likely to be local (probably at the active site) and, unlike stability, not a global feature of the enzyme. Therefore, it will be important when assessing the effect of engineered mutations on enzymes to determine the effects on stability and activity separately. This might involve a compromise – Teq can be increased by mutations that increase DHeq and/or decrease DSeq. The former might be easier to achieve through the introduction of stabilizing interactions at the active site, which might reduce the flexibility of the protein and hence its tendency to deform as the temperature is raised. However, given that conformational flexibility is often required for efficient catalysis, it might turn out to be difficult to alter Teq using such a mutational strategy without adversely affecting the kcat value of the enzyme. An understanding of the molecular basis of the equilibrium between Eact and Einact would be desirable, particularly to guide and interpret mutagenesis experiments. NMR analysis of the enzyme structure over its temperature range of catalytic activity is one possibility, although significant irreversible thermal unfolding of the protein during the time required to collect the spectra might be a problem if Teq and Tm were close in value.

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Figure 3. Effect of Teq on time courses of product concentration simulated at various temperatures: 310 K (blue); 320 K (green); 330 K (purple); 340 K (red). (a) Equilibrium Model with TeqZ300 K; (b) Equilibrium Model with TeqZ350 K; (c) Classical Model for comparison. The other parameter values are the same as those used in Figure 1.

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Enzyme reactors Maximising the output of enzyme reactors requires a careful balance of the effects of temperature on enzyme stability and activity. Predictions of the output of product with time and temperature based on the Classical Model can be different from those derived from the Equilibrium Model. When using an enzyme in a batch reactor for chemical synthesis, intuition would predict that the higher the operating temperature, the faster the catalysed reaction but also the less stable the enzyme. In fact, this is only generally true if Teq exceeds the working temperature of the reactor. If Teq is less than the working temperature, the reverse is true (compare Figure 3a with 3b). Typical time courses of product formation at various temperatures, predicted by the Classical Model, which will never show the effect seen in Figure 3a, are shown in Figure 3c for comparison. Enzyme screening and selecting for applications Enzyme screening or selection for particular thermal properties is normally carried out to obtain good activity and/or resistance to denaturation at a particular temperature. Enzyme thermal stability is usually correlated with the growth temperature of the source organism: enzymes from thermophiles are generally more thermally stable than those from mesophiles, whereas those from psychrophiles are relatively less stable. Exceptions to this are known, although we would expect Teq to be even more closely associated with the growth temperature of the cell than thermal stability. The simple Classical Model for the thermal behaviour of enzymes suggests that both activity and resistance to denaturation can be achieved by assessment of thermal stability alone. The Equilibrium Model shows that the reduction of activity as the temperature is raised might not be due to irreversible thermal inactivation – stability is necessary for activity but does not guarantee it. Therefore, to gain the full benefit from screening, both stability and Teq must be sought or selected separately. In short, successful selection from the environment of

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thermally stable and/or active enzymes will depend upon making a clear distinction between activity at high temperatures (determined by Teq) and thermal stability. Acknowledgements We are grateful to the New Zealand Marsden and ISAT Linkages Funds for financial support.

References 1 Schiraldi, C. and De Rosa, M. (2002) The production of biocatalysts and biomolecules from extremophiles. Trends Biotechnol. 20, 515–521 2 Adams, M.W.W. and Kelly, R.M. (1998) Finding and using hyperthermophilic enzymes. Trends Biotechnol. 16, 329–332 3 Coolbear, T. et al. (1992) The enzymes from extreme thermophiles: bacterial sources, thermostabilities and industrial relevance. Adv. Biochem. Eng. Biotechnol. 45, 57–98 4 Cowan, D.A. (1997) Thermophilic proteins: stability and function in aqueous and organic solvents. Comp. Biochem. Physiol. A Physiol 118, 429–438 5 Daniel, R.M. et al. (1982) A correlation between protein thermostability and resistance to proteolysis. Biochem. J. 207, 641–644 6 Daniel, R.M. et al. (2001) The temperature optima of enzymes: a new perspective on an old phenomenon. Trends Biochem. Sci. 26, 223–225 7 Daniel, R.M. and Danson, M.J. (2001) Assaying activity and assessing thermostability of hyperthermophilic enzymes. Methods Enzymol. 334, 283–293 8 Cornish-Bowden, A. (2004) Chapter 10: Temperature effects on enzyme activity. In Fundamentals of Enzyme Kinetics (3rd Edn), pp. 231–238, Portland Press Ltd 9 Thomas, T.M. and Scopes, R.K. (1998) The effects of temperature on the kinetics and stability of mesophilic and thermophilic 3-phosphoglycerate kinases. Biochem. J. 330, 1087–1095 10 Gerike, U. et al. (1997) Sequencing and expression of the gene encoding a cold-active citrate synthase from an Antarctic bacterium, strain DS2–3R. Eur. J. Biochem. 248, 49–57 11 Peterson, M.E. et al. (2004) A new intrinsic thermal parameter for enzymes reveals true temperature optima. J. Biol. Chem. 279, 20717–20722 12 Tsou, C.L. (1995) Inactivation precedes overall molecular-conformation changes during enzyme denaturation. Biochim. Biophys. Acta 1253, 151–162 13 Sizer, I.W. (1944) Temperature activation and inactivation of the crystalline catalase–hydrogen peroxide system. J. Biol. Chem. 154, 461–473 14 Wright, G.G. and Schomaker, V.J. (1948) Studies on the denaturation of antibody. iii. Kinetic aspects of the inactivation of diphtheria antitoxin by urea. Am. Chem. Soc. 70, 356–364

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