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Thermodynamic Analysis of Catalysis by the Dihydroorotases from Hamster and Bacillus caldolyticus, As Compared with the Uncatalyzed Reaction^†

Danny T. Huang,^‡ Jacob Kaplan,^§ R. Ian Menz, Vittorio L. Katis, R. Gerry Wake,^§ Feng Zhao,^# Richard Wolfenden,^# and Richard I. Christopherson*^§

School of Molecular and Microbial Biosciences, University of Sydney, Sydney, New South Wales 2006, Australia, and Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260

Biochemistry, 2006, 45 (27), pp 8275–8283

DOI: 10.1021/bi060595w

Publication Date (Web): June 15, 2006

†

This work was supported by the Australian National Health and Medical Research Council, Project Grant 253781.

‡

Present address: Department of Structural Biology and Genetics/Tumor Cell Biology, St. Jude's Children's Research Hospital, Memphis, TN 38105.

University of Sydney.

Present address: School of Biological Sciences, Flinders University of South Australia, GPO Box 2100, Adelaide, SA 5001, Australia.

Present address: Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, U.K.

University of North Carolina.

To whom correspondence should be addressed. Phone: 61-2-9351-6031. Fax: 61-2-9351-4726. E-mail: ric@mmb.usyd.edu.au. URL: http://www.mmb.usyd.edu.au.

Abstract

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher V_max and K_s values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of l-dihydroorotate were determined at 25 °C for hamster DHOase (ΔG = −6.9 kcal/mol, ΔH = −11.5 kcal/mol, TΔS = −4.6 kcal/mol) and B. caldolyticus DHOase (ΔG = −5.6 kcal/mol, ΔH = −4.2 kcal/mol, TΔS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the k_cat/K_s value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 °C to 5.3 kcal/mol for temperatures of 50−70 °C. Vibrational energy at higher temperatures may facilitate the transition ES → ES, making k_cat/K_s almost temperature independent. The pseudo-first-order rate constant for water attack on l-dihydroorotate, based on experiments at elevated temperature, is 3.2 × 10^-¹¹ s^-¹ at 25 °C, with ΔH = 24.7 kcal/mol and TΔS = −6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 × 10¹⁴, achieving this rate enhancement almost entirely by lowering the enthalpy of activation (ΔΔH = −19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme−substrate complex in the ground state.